Methods for killing antibiotic tolerant bacteria

ABSTRACT

Disclosed herein are compounds, compositions, and methods for treating infections by bacteria and for killing bacteria. In particular, the disclosed compounds, compositions, and methods are useful for treating infections by bacteria and for killing persister bacteria that survive antibiotic treatment and then reconstitute infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e)to U.S. Provisional Application No. 63/033,544, filed Jun. 2, 2020, thecontent of which is incorporated herein by reference in its entirety

BACKGROUND

The field of the invention relates to compounds, compositions, andmethods for reduction and eradication of persister cells in a bacterialpopulation. More specifically, the field of the invention relates tocompounds, compositions, and methods for treating bacterial infections.

Bacterial “persister cells” are pathogenic and nonpathogenic bacteriathat neither grow nor die in the presence of microbicidal antibiotics.As such, persister cells contribute to the recalcitrance of treatment ofclinical infections by reconstituting infections. Persister cells arisedue to metabolic inactivity. (See K. Lewis, Nat. Rev. Microbiol. 5, 48(2007); Kwan, et al. Antimicrob. Agents Chemother. 57, 1468 (2013); andT. K. Wood, et al., Appl. Environ. Microbiol. 79, 7116 (2013)).Persister cells are highly tolerant to all traditional antibiotics,which are primarily effective against actively growing cells, as well astolerant to other stresses such as lack of nutrients, oxidizing agents,acids, bases, and temperature extremes.

Bacterial persistence is a non-hereditary phenotype that may occurstochastically or through environmental influence in a smallsub-population of all tested bacterial species. (See W. Bigger, Lancet244, 497 (1944); N. Q. Balaban, et al., Science 305, 1622 (2004); Kwan,et al. (2013), N. Moker, et al., J. Bacteriol. 192, 1946 (2010); Y. Hu,et al., Environ. Microbiol., 17, 1275, (2015); T. Dorr, et al. PLoSBiol. 8, e1000317 (2010); and N. M. Vega, et al., Nat. Chem. Biol. 8,431 (2012); and K. Lewis, Curr. Top. Microbiol. Immunol. 322, 107(2008)). Few distinctly new antibiotics have been discovered recently.(See T. J. Dougherty, M. J. Pucci, Eds., (Springer, New York, N.Y.,2012). Worse, current antibiotics are ineffective against persistercells. Thus, there is an ongoing and unmet need for improved approachesto treating infections that comprise persister cells. The presentdisclosure meets this need.

SUMMARY

Disclosed herein are compounds, compositions, and methods for treatinginfections by bacteria and for killing bacteria. In particular, thedisclosed compounds, compositions, and methods are useful for treatinginfections by bacteria and for killing persister bacteria that surviveantibiotic treatment and then reconstitute infections.

The disclosed compounds, compositions, and methods may be utilized forreducing and/or eradicating bacterial persister cells and/or dormant“viable but non-culturable” (VBNC) cells. The disclosed methodstypically comprise administering the disclosed compounds or compositionscomprising the disclosed compounds to a bacterial population comprisingbacterial persister cells and/or dormant VBNC cells where preferably thebacterial persister cells and/or the VBNC cells of the bacterialpopulation are killed, reduced, and/or eradicated. The disclosedcompounds for use in the disclosed compositions and methods may include,but are not limited to [(5-nitro-3-phenyl-1H-indol-2-yl)methyl]amine andsalts thereof such as [(5-nitro-3-phenyl-1H-indol-2-yl)methyl]aminehydrochloride (NPIMA), and2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one(BPOET) or salts thereof.

The disclosed compounds, compositions, and methods may be utilized forkilling, reducing, and/or eradicating bacteria of a variety of types,including Gram-negative and Gram-positive pathogenic bacteria. Incertain approaches, the bacteria are pathogenic and/or are selected fromEscherichia spp. such as enterohemorrhagic E. coli, Staphylococcus spp.such as S. aureus, and Pseudomonas spp. such as Pseudomonas aeruginosa.

The disclosed compounds, compositions, and methods may be utilized forkilling, reducing, and/or eradicating bacteria from a wound of anindividual, optionally wherein the wound comprises bacteria that areresistant to one or more antibiotics. Optionally, the individual mayhave been previously been diagnosed with a bacterial infection andtreated with at least one antibiotic, for example, where the bacterialinfection was not cleared by such previous treatment.

In the disclosed methods, the bacterial population may be present in aliquid biological sample or liquid environment. In some embodiments ofthe disclosed methods, the bacterial population is present insuspension.

The disclosed compounds, compositions, and methods may be utilized forkilling, reducing, and/or eradicating bacteria in a biofilm, optionallywherein the bacteria are present in a biofilm and are killed, reduced,and/or eradicated, and preferably the biofilm is not dispersed. In someembodiments, the bacteria may be present on an inanimate surface,including but not necessarily limited to a medical device, including butnot necessarily limited to implantable or implanted medical devices.

The disclosed compounds, compositions, and methods may be utilized forkilling, reducing, and/or eradicating bacteria in aggregates, optionallywherein the bacteria are present in aggregates and are killed, reduced,and/or eradicated, and preferably the aggregates are not dispersed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . Most effective persister killing compounds identified fromscreening persister cells. Escherichia coli persister cells were treatedfor 24 hr at 100 µM with:

-   1: N-(3,4-dichlorophenyl)-N′-(3-fluorophenyl) thiourea;-   2: 2-({2-[(4-bromophenyl)amino]-4-quinazolinyl}amino)ethanol    hydrochloride;-   3: 2-[(6-phenyl-2,3,4,9-tetrahydro-1H-carbazol-1-yl)amino] ethanol;-   4:    1-(3,6-dichloro-9H-carbazol-9-yl)-3-(2-methyl-1H-imidazol-1-yl)-2-propanol;-   5: N-[2-(4-fluorophenyl)ethyl]-N′-(4-nitrophenyl) thiourea;-   6: N-(4-chlorobenzyl)-N′-4-pyridinylthiourea;-   7: N′-(3,5-dichloro-    2-hydroxybenzylidene)-2-oxo-4-phenyl-3-pyrrolidinecarbohydrazide;    and-   8: [(5-nitro-3-phenyl-1H-indol-2-yl)methyl]amine hydrochloride    (NPIMA, structure shown in inset).

FIG. 2 . NPIMA eradicates Escherichia coli, Pseudomonas aeruginosa, andStaphylococcus aureus exponential cells. Survival after 6 hr for (a) E.coli BW25113 with NPIMA and 5-iodoindole at 100 µM, (b) P. aeruginosaPA14 with NPIMA and 5-iodoindole at 100 µM, and (c) S. aureus with NPIMAand 5-iodoindole at 200 µM. Asterisk indicates no viable cells detected.NPIMA, 5-nitro-3-phenyl-1H-indol-2-yl-methylamine hydrochloride.

FIG. 3 . NPIMA kills Escherichia coli, Pseudomonas aeruginosa, persistercells. Survival of (a) E. coli BW25113 and (b) P. aeruginosa PA14 aftertreatment with NPIMA (100 µM), 5-iodoindole (5-II, 100 µM), and DMSO for6 hr. Asterisk indicates no viable cells detected. DMSO, dimethylsulfoxide; NPIMA, 5-nitro-3-phenyl-1H-indol-2-yl-methylaminehydrochloride.

FIG. 4 . NPIMA lyses Escherichia coli, Pseudomonas aeruginosa, andStaphylococcus aureus. LIVE/DEAD staining of (a) E. coli BW25113 after0.75 hr, (b) P. aeruginosa PA14 after 1 hr, and (c) S. aureus after 1 hrtreatment with NPIMA at 100 µM. Left shows dark field, middle shows allcells stained by Syto9, and right shows dead cells stained by propidiumiodide. White arrows indicate lysed cells. Representative images areshown. Scale bar = 10 µM. NPIMA,5-nitro-3-phenyl-1H-indol-2-ylmethylamine hydrochloride.

FIG. 5 . NPIMA lyses Escherichia coli, Pseudomonas aeruginosa, andStaphylococcus aureus. Total protein (a) and DNA (b) found insupernatants as evidence of cell lysis after treatment of cells at aturbidity of 0.8 at 600 nm with 100 µM NPIMA after 1 hr for E. coli andP. aeruginosa and 6 hr for S. aureus. NPIMA,5-nitro-3-phenyl-1H-indol-2-yl-methylamine hydrochloride.

FIG. 6 . NPIMA damages the Escherichia coli cell membrane. Transmissionelectron microscopic (TEM) images of E. coli BW25113 after NPIMA (100µM) treatment for 40 min. Two representative images are shown. The arrowindicates membrane damage. NPIMA,5-nitro-3-phenyl-1H-indol-2-ylmethylamine hydrochloride.

FIG. 7 . NPIMA compared width cisplatin for killing Pseudomonasaeruginosa, Escherichia coli, and Staphylococcus aureus. Survival ofexponentially-growing cells of P. aeruginosa PA14 (a), E. coli (b), andS. aureus (c) treated with one MIC of NPIMA (♦) and cisplatin (•) for 3hr in PBS. DMSO (▲) and NaClO4 (*) were used as a negative controls.MIC, minimum inhibitory concentration; NPIMA,5-nitro-3-phenyl-1H-indol-2-yl-methylamine hydrochloride.

FIG. 8 . NPIMA structure function relationships. Exponential cells ofEscherichia coli BW25113 were treated with (a) 2-(aminomethyl) indole,and (b) 2-methyl-5-nitro-3-phenyl-1H-indole for 6 hr. NPIMA,5-nitro-3-phenyl-1Hindol-2-yl-methylamine hydrochloride.

FIG. 9 . NPIMA kills naturally-occurring E. coli persister cells.Survival of E. coli with 5-iodoindole and solvent negative control(DMSO) also shown. Asterisk (*) indicates no viable cells were detected.The persister cells were harvested at stationary-phase of E. coli(turbidity of 6.0 at 600 nm) and non-persister cells were removed bytreatment.

FIG. 10 . NPIMA kills S. aureus and P. aeruginosa in a wound model.Survival of total cells ( P. aeruginosa + S. aureus) in the wound modelafter treatment with NPIMA for 6 h. The left bar is the solvent (DMSO)negative control.

FIG. 11 . Cytotoxicity of NPIMA with HT-29 cells. (A) Cell viability wasmeasured via a CCK-8 assay. (B) Cytotoxicity was measured with variousconcentrations of NPIMA (0, 5, to each well along with - in 98) for 24h. Cell viability was measured at 450 nm, and cell toxicity was measuredat 492 nm and a reference wavelength (620 nM) was used.

FIG. 12 . RluD increases persister resuscitation by increasing ribosomesfor resuscitation. (A) Single-cell persister resuscitation as determinedusing light microscopy (Zeiss Axio Scope.A1). The total and wakingnumber of persister cells are shown in Table 5. Microscope images forwaking cells are shown in FIG. 13 . The fold-change in resuscitation isrelative to BW25113 with DMSO for BW25113 with BPOET, relative toBW25113 for the ΔrluD mutant, relative to BW25113/pCA24N for the strainproducing RluD from pCA24N plasmid in BW25113, and relative to MG1655for ΔrybB. M9 glucose (0.4%) agarose gel pads were used for all thestrains except BW25113 with BPOET where M9 alanine (5X) agarose gel padsincluding 100 µM of BPOET or DMSO were used. The results are thecombined observations from two independent experiments after 6 h for theBW25113 with BPOET, after 4 h for BW25113 and its deletion mutants, andafter 6 h for cells harboring pCA24N and its derivatives as well as forMG1655 and MG1655 ΔrybB. Error bars indicate standard deviations. (B)Active 70S ribosomes in single persister cells forMG1655-ASV/pCA24N-rluD (“RluD”) vs. MG1655-ASV/pCA24N (“Empty”). Cellsare shown on agarose gel pads at time 0 for resuscitation; i.e., afterthe formation of persister cells. Representative results from threeindependent cultures are shown.

FIG. 13 . Single persister cell waking. Persister cells of (A) BW25113with DMSO (upper panel), and BPOET (lower panel) on M9 5X Ala agarosegel pads containing DMSO and BPOET (100 µM) after 6 h, (B) BW25113(upper panel) and BW25113 ΔrluD (lower panel) after 4 hours on M9 0.4%glucose agarose gel pads, (C) BW25113/pCA24N (“Empty”), andBW25113/pCA24N-rluD (“RluD”) after 6 h on M9 0.4% glucose agarose gelpads, and (D) MG1655, and MG1655 ΔrybB after 6 h on M9 0.4% glucoseagarose gel pads. Arrows indicate cells that resuscitate. Scale barindicates 10 µm. Representative results from two independent culturesare shown.

FIG. 14 . Inactivating RluD eliminates persister cell waking on minimalglucose medium but does not affect the number of persister cells thatare formed. (A) Resuscitation of wild type BW25113 and BW25113 ΔrluDpersister at 37° C. on M9 0.4% glucose agar plates for three days. (B)Colonies formed in one day at 37° C. on LB agar plates indicating thenumber of persister cells for BW25113 and the isogenic ΔrluD mutant. Onerepresentative plate of two independent cultures is shown.

DETAILED DESCRIPTION

The present invention is described herein using several definitions, asset forth below and throughout the application.

Definitions

Unless otherwise specified or indicated by context, the terms “a”, “an”,and “the” mean “one or more.” For example, “a compound” or “anantibiotic” should be interpreted to mean “one or more compounds” and“one or more antibiotics,” respectively.

As used herein, “about,” “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of these terms which are not clear to persons ofordinary skill in the art given the context in which they are used,“about” and “approximately” will mean plus or minus ≤10% of theparticular term and “substantially” and “significantly” will mean plusor minus >10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising” in that these latterterms are “open” transitional terms that do not limit claims only to therecited elements succeeding these transitional terms. The term“consisting of,” while encompassed by the term “comprising,” should beinterpreted as a “closed” transitional term that limits claims only tothe recited elements succeeding this transitional term. The term“consisting essentially of,” while encompassed by the term “comprising,”should be interpreted as a “partially closed” transitional term whichpermits additional elements succeeding this transitional term, but onlyif those additional elements do not materially affect the basic andnovel characteristics of the claim.

As used herein, a “subject” may be interchangeable with “patient” or“individual” and means an animal, which may be a human or non-humananimal, in need of treatment, for example, treatment by includeadministering a therapeutic amount of one or more compounds for killing,reducing, and/or eradicating bacteria from a bacterial population. A“subject in need of treatment” may include a subject having an infectioncharacterized by the presence of bacterial persister cells and/ordormant viable but non-culturable (VBNC) cells.

The present disclosure provides methods for killing, reducing, and/oreradicating bacterial persister cells and/or dormant viable butnon-culturable (VBNC) cells in a bacterial population. The persisterand/or VBNC cells may be present in population that comprises, consistsessentially of, or consists of such cells. The disclosed methodstypically comprise administering an effective amount of a compound ofTable 1 or Table 4 (or a non-salt or salt form thereof), wherein thebacterial persister cells and/or the VBNC cells in the bacterialpopulation are killed, reduced, and/or or eradicated. Suitable compoundsinclude, but are not limited to[(5-nitro-3-phenyl-1H-indol-2-yl)methyl]amine and salts thereof such as[(5-nitro-3-phenyl-1H-indol-2-yl)methyl]amine hydrochloride (NPIMA), and2-{[2-(4-bromophenyl)-2-oxoethyl]}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one(BPOET) or salts thereof.

As used herein, the term “compound” includes any compound listed inTable 1 or Table 4, or salt forms or non-salt forms thereof. Inaddition, where the compound listed in Table 1 or Table 4 is a saltform, such as NPIMA which is a hydrochloride salt of[(5-nitro-3-phenyl-1H-indol-2-yl)methyl]amine, the term “compound”includes alternative salt forms of the compound listed in Table 1 orTable 4. An alternative salt form of NPIMA may include a salt form thatis not a hydrochloride salt form.

“Persister cells” as used herein means bacteria, including pathogenicbacteria, that neither grow nor die in the presence of microbicidalantibiotics with the exception of the compounds disclosed herein.“Persister cells” as used herein also include antibiotic tolerant cells.The disclosed compounds, compositions, and methods may be utilized forkilling persister cells and/or reducing or eradicating the number ofpersister cells in a bacterial population. The population of persistercells can be present in an infection (e.g., in a subject), in abiological sample, or on an inanimate surface. In nonlimiting examples,surfaces may include non-porous surfaces, such as surfaces in ahospital, surfaces on a medical device, and/or a surfaces that are usedfor food processing or preparation.

Persister cells and/or VBNC cells that are killed, reduced, and/oreradicated may be responsible for and/or positively correlated with thepresence of recalcitrant infections, such as chronic recalcitrantinfections. Chronic recalcitrant infections are known to occur when thefunctioning of an individual’s immune system is less than optimal. Knownexamples of chronic infection occur in a variety of individuals, such asthose who are immunocompromised because of immunosuppressive drugcourses, co-infection with viruses, or individuals who have an infectionthat forms a biofilm or aggregates. “Biofilm” as used herein is anassemblage of surface-associated microbial cells that is enclosed in anextracellular polymeric substance matrix. “Biofilm” as used herein alsois an aggregate of free-floating cells. As is also well known in theart, biofilms and aggregates impede access of immune cells andimmunological signaling molecules to bacteria, and thus limit theeffectiveness of even normally functioning immune systems. Further,biofilms are known to form in a variety of wounds inside the body, aswell as on surfaces of indwelling medical devices. In certain instances,such infections and biofilms can be populated by drug-resistant bacteriapresent on the devices, and in the tissue that comes into contact withthem. However, in many instances chronic and recalcitrant infectionsarise because comparatively slow-growing bacteria develop intodrug-tolerant persister cells that are difficult to eradicate withcurrently used antibiotics, and this can occur with or without thepresence of an implanted device. Thus, upon cessation of a course ofantibiotics and the subsequent decrease in its concentration, persistercells can exploit an opportunity to grow and repopulate the infectionand/or biofilm. The approaches of the present disclosure areparticularly suited for reducing the number of and/or eradicating suchcells.

In some embodiments, the methods disclosed herein may be utilized toreduce persister cells and/or VBNC cells present in a bacterialpopulation, wherein the reduction is greater than a reference. Thereference may comprise any suitable control, value or measurement of thereduction of persister cells, such as a standardized curve, a titration,the area under a curve, or a comparison to the capability of anotherantimicrobial compound to kill the persister cells and/or the VBNCcells. In some embodiments, the reference comprises a value obtainedfrom measuring the amount of persister cells and/or VBNC cells of thesame bacterial species that are killed using an antibiotic that is not acompound of Table 1 or Table 4 (or a salt or non-salt thereof). In someembodiments, the amount of the reference antibiotic used to compare tocompound of Table 1 or Table 4, or a salt or non-salt thereof, i.e., anamount of the reference antibiotic that kills non-persistent cells atthe same minimum inhibitory concentration (MIC) of compound of Table 1or Table 4 (or a salt or non-salt thereof), respectively.

The disclosed compositions for killing, reducing, and/or eradicatingpersister cells may comprise, consist essentially of, or consist of acompound of Table 1 or Table 4 (or a salt or non-salt thereof) such asNPIMA and BPOET, preferably for use as an antibiotic. In someembodiments, the disclosed compositions comprise, consist essentiallyof, or consist of Table 1 or Table 4 (or a salt or non-salt thereof), asthe only antibiotic agent in the composition. In other embodiments, thedisclosed composition comprise, consist essentially of, or consist oftwo or more antibiotic agents including at least a compound of Table 1or Table 4 (or a salt or non-salt thereof) and further including anantibiotic that that is a member of classes such as aminoglycosides,beta lactams (with or without beta lactamase inhibitor such asclavulanic acid), macrolides, glycopeptides, polypeptides,cephalosporins, lincosamides, ketolides, rifampicin, polyketides,carbapenem, pleuromutilin, quinolones, streptogranins, oxazolidinones,lipopeptides, and the like.

In the disclosed methods, a bacterial population may be administered acompound of Table 1 or Table 4 (or a salt or non-salt thereof) as anantibiotic and the bacterial population further may be administered oneor more different antibiotics which may be a compound of Table 1 orTable 4 (or a salt or non-salt thereof) or an antibiotic that that is amember of classes such as aminoglycosides, beta lactams (with or withoutbeta lactamase inhibitor such as clavulanic acid), macrolides,glycopeptides, polypeptides, cephalosporins, lincosamides, ketolides,rifampicin, polyketides, carbapenem, pleuromutilin, quinolones,streptogranins, oxazolidinones, lipopeptides, and the like. As such, thebacterial population may be administered two or more antibiotics, whichmay be administered concurrently or sequentially, where one antibioticis administered to the bacterial population and another antibiotic issubsequently administered to the bacterial population.

In some embodiments of the disclosed methods, a first antibiotic isadministered to a bacterial population, which first antibiotic is amember of classes such as aminoglycosides, beta lactams (with or withoutbeta lactamase inhibitor such as clavulanic acid), macrolides,glycopeptides, polypeptides, cephalosporins, lincosamides, ketolides,rifampicin, polyketides, carbapenem, pleuromutilin, quinolones,streptogranins, oxazolidinones, lipopeptides, and the like, andsubsequently a second antibiotic is administered to the bacterialpopulation, which second antibiotic is a compound of Table 1 or Table 4(or a salt or non-salt thereof) is administered. In other embodiments, afirst antibiotic is administered to a bacterial population, which firstantibiotic is a compound of Table 1 or Table 4 (or a salt or non-saltthereof) is administered, and subsequently a second antibiotic isadministered to the bacterial population, which second antibiotic is amember of classes such as aminoglycosides, beta lactams (with or withoutbeta lactamase inhibitor such as clavulanic acid), macrolides,glycopeptides, polypeptides, cephalosporins, lincosamides, ketolides,rifampicin, polyketides, carbapenem, pleuromutilin, quinolones,streptogranins, oxazolidinones, lipopeptides, and the like.

In some embodiments of the disclosed methods, the persister cells and/orVBNC cells may be resistant to one or more antibiotics. For example, insome embodiments of the disclosed methods, the persister cells and/orVBNC cells may be resistant to an antibiotic which is a member ofclasses such as aminoglycosides, beta lactams (with or without betalactamase inhibitor such as clavulanic acid), macrolides, glycopeptides,polypeptides, cephalosporins, lincosamides, ketolides, rifampicin,polyketides, carbapenem, pleuromutilin, quinolones, streptogranins,oxazolidinones, lipopeptides, and the like.

Various methods known to those skilled in the art may be used toadminister the disclosed compounds and compositions comprising thedisclosed compounds for the purpose of reducing or eradicatingpopulations of persister cells and/or VBNC cells, including suchpopulations when they are present in an infection in an individual.These methods include but are not necessarily limited to intradermal,transdermal, intravenous, topical, intramuscular, intraperitoneal,intravenous, subcutaneous, oral, and intranasal routes. In certainaspects the disclosure includes providing the compounds in the form ofcreams, aqueous solutions, suspensions or dispersions, oils, balms,foams, lotions, gels, cream gels, hydrogels, liniments, serums, films,ointments, sprays or aerosols, other forms of coating, or any multipleemulsions, slurries or tinctures. The compositions may be embedded inmaterials, such as a medical device or other implement used in treatingor manipulating a body, organ, tissue or biological fluid. Thecompositions can also include liposomes, microsomes, nanoparticles, andany other suitable vehicle for delivering the compounds such that theyreduce or eradicate persister cells where present. Further, it will berecognized by those of skill in the art that the form and character ofthe particular dosing regimen employed in the method of this disclosurewill be dictated by the route of administration and other well-knownvariables, such as the age, sex, health and size of the individual, thetype and severity of bacterial infection, or risk of bacterialinfection, and other factors that will be apparent to the skilledartisan given the benefit of the present disclosure. improve the time inblood circulation, the disclosed compounds and compositions comprisingthe disclosed compounds may be combined with nano-formulations comprisedof microemulsions, carbon nanoparticles, true nano-spheres, orpolyanionic PEG-polyglutamate co-polymers.

In certain embodiments, the method of the disclosure results ineradication of a bacterial population comprising the bacterial persistercells and/or VBNC cells from an infection, such as from an infection ofan organ, tissue, skin, or biological fluid from an individual, or fromthe surface of an inanimate object, including but not necessarilylimited to medical devices, such as implantable or implanted medicaldevices, and/or any medical device that may stay in contact with theskin or be fully or partially present within the body of an individualfor a period of time during which the surface of the device may besusceptible to biofilm formation.

The disclosed compounds, compositions, and methods may be utilized forkilling, reducing, and/or eradicating bacteria that are present inanaerobic conditions. The disclosed compounds, compositions, and methodsmay be utilized for killing, reducing, and/or eradicating bacteria of avariety of types, include Gram-negative and Gram-positive pathogenicbacteria. In certain approaches, the bacteria are pathogenic and/or areselected from Escherichia spp. such as E. coli, Staphylococcus spp. suchas S. aureus, and Pseudomonas spp. such as Pseudomonas aeruginosa. Inother embodiments, disclosed compounds, compositions, and methods may beutilized for killing, reducing, and/or eradicating bacteria that areselected from V. cholerae, B. burgdorferi, Streptococcus spp., S.typhimurium, E. faecalis, A. baumannii, A. iwoffii, S. marcescens, P.mirabilis, K. pneumoniae, A. calcoaceticus, S. mutans, P. gingivalis, H.influenza, H. pylori, N. meningitides, N. gonorrhea, M. kansasii, B.anthracis, P. acnes, C. tetani, C. trachomatis, L. pneumophila, Y.pestis, B. abortus, F. tularensis, V. harveyi, and combinations thereof.

In some embodiments, the disclosed compounds, compositions, and methodsmay be utilized for killing, reducing, and/or eradicating bacteria thatare present in a wound of an individual. In some embodiments, thedisclosed methods comprise administering to a subject in need thereof acomposition comprising an effective amount of a compound of Table 1 orTable 4 (or a salt or non-salt form thereof). A subject in need thereofincludes a subject known to be in need of reducing or eradicating aninfection, including an infection comprising persister cells and/or VBNCcells.

In some embodiments, the disclosed compounds, compositions, and methodsmay be utilized for killing, reducing, and/or eradicating bacteria thatare present in a liquid biological sample or liquid environment. Forexample, the disclosed compounds, compositions, and methods may beutilized for killing, reducing, and/or eradicating bacteria that arepresent in suspension.

In some embodiments, the disclosed compounds, compositions, and methodsmay be utilized to kill, reduce, and/or eradicate persister cells and/orVBNC cells in an infection in a subject who has been diagnosed with abacterial infection and has been treated with at least one antibioticother than the compounds disclosed in Table 1 and Table 4 (or salt ornon-salt forms thereof), optionally where the diagnosed bacterialinfection was not cleared by the previous treatment. In someembodiments, the subject has been treated with an antibiotic that is amember of classes such as aminoglycosides, beta lactams (with or withoutbeta lactamase inhibitor such as clavulanic acid), macrolides,glycopeptides, polypeptides, cephalosporins, lincosamides, ketolides,rifampicin, polyketides, carbapenem, pleuromutilin, quinolones,streptogranins, oxazolidinones, lipopeptides, and the like.

In some embodiments, the disclosed compounds, compositions, and methodsmay be utilized to kill, reduce, and/or eradicate persister cells and/orVBNC cells that are present in a biofilm. In some embodiments, thebacterial persister cells and/or VBNC cells are reduced or eradicatedfrom a biofilm, but the biofilm is not dispersed.

In some embodiments, the disclosed compounds, compositions, and methodsmay be utilized to kill, reduce, and/or eradicate persister cells and/orVBNC cells that may be present on an inanimate surface. Inanimatesurfaces may include, but are not limited to surfaces present on animplanted medical device or surfaces outside a body.

The compounds disclosed herein may be administered as pharmaceuticalcompositions and, therefore, pharmaceutical compositions incorporatingthe compounds are considered to be embodiments of the subject matterdisclosed herein. Such compositions may take any physical form which ispharmaceutically acceptable; illustratively, they can be orallyadministered pharmaceutical compositions. Such pharmaceuticalcompositions contain an effective amount of a disclosed compound, whicheffective amount is related to the daily dose of the compound to beadministered. Each dosage unit may contain the daily dose of a givencompound or each dosage unit may contain a fraction of the daily dose,such as one-half or one-third of the dose. The amount of each compoundto be contained in each dosage unit can depend, in part, on the identityof the particular compound chosen for the therapy and other factors,such as the indication for which it is given. The pharmaceuticalcompositions disclosed herein may be formulated so as to provide quick,sustained, or delayed release of the active ingredient afteradministration to the patient by employing well known procedures.

As indicated above, pharmaceutically acceptable salts of the compoundsare contemplated and also may be utilized in the disclosed methods. Theterm “pharmaceutically acceptable salt” as used herein, refers to saltsof the compounds which are substantially non-toxic to living organisms.Typical pharmaceutically acceptable salts include those salts preparedby reaction of the compounds as disclosed herein with a pharmaceuticallyacceptable mineral or organic acid or an organic or inorganic base. Suchsalts are known as acid addition and base addition salts. It will beappreciated by the skilled reader that most or all of the compounds asdisclosed herein are capable of forming salts and that the salt forms ofpharmaceuticals are commonly used, often because they are more readilycrystallized and purified than are the free acids or bases.

Acids commonly employed to form acid addition salts may includeinorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, phosphoric acid, and the like, and organic acidssuch as p-toluenesulfonic, methanesulfonic acid, oxalic acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of suitablepharmaceutically acceptable salts may include the sulfate, pyrosulfate,bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide,acetate, propionate, decanoate, caprylate, acrylate, formate,hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate,propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate,maleat-, butyne-.1,4-dioate, hexyne-1,6-dioate, benzoate,chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate,phthalate, xylenesulfonate, phenylacetate, phenylpropionate,phenylbutyrate, citrate, lactate, alpha-hydroxybutyrate, glycolate,tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate, and the like.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Bases useful in preparing such salts includesodium hydroxide, potassium hydroxide, ammonium hydroxide, potassiumcarbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate,calcium hydroxide, calcium carbonate, and the like.

The particular counter-ion forming a part of any salt of a compounddisclosed herein is may not be critical to the activity of the compound,so long as the salt as a whole is pharmacologically acceptable and aslong as the counterion does not contribute undesired qualities to thesalt as a whole. Undesired qualities may include undesirably solubilityor toxicity.

Pharmaceutically acceptable esters and amides of the compounds can alsobe employed in the compositions and methods disclosed herein. Examplesof suitable esters include alkyl, aryl, and aralkyl esters, such asmethyl esters, ethyl esters, propyl esters, dodecyl esters, benzylesters, and the like. Examples of suitable amides include unsubstitutedamides, monosubstituted amides, and disubstituted amides, such as methylamide, dimethyl amide, methyl ethyl amide, and the like.

In addition, the methods disclosed herein may be practiced using solvateforms of the compounds disclosed herein or salts, esters, and/or amides,thereof. Solvate forms may include ethanol solvates, hydrates, and thelike.

As used herein, the terms “treating” or “to treat” each mean toalleviate symptoms, eliminate the causation of resultant symptoms eitheron a temporary or permanent basis, and/or to prevent or slow theappearance or to reverse the progression or severity of resultantsymptoms of the named disease or disorder. As such, the methodsdisclosed herein encompass both therapeutic and prophylacticadministration.

As used herein, the phrase “effective amount” shall mean that drugdosage that provides the specific pharmacological response for which thedrug is administered in a significant number of subjects in need of suchtreatment. An effective amount of a drug that is administered to aparticular subject in a particular instance will not always be effectivein treating the conditions/diseases described herein, even though suchdosage is deemed to be a therapeutically effective amount by those ofskill in the art.

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount or dose of compound administered, anumber of factors can be considered by the attending diagnostician, suchas: the species of the subject; its size, age, and general health; thedegree of involvement or the severity of the disease or disorderinvolved; the response of the individual subject; the particularcompound administered; the mode of administration; the bioavailabilitycharacteristics of the preparation administered; the dose regimenselected; the use of concomitant medication; and other relevantcircumstances.

A typical daily dose may contain from about 0.01 mg/kg to about 100mg/kg (such as from about 0.05 mg/kg to about 50 mg/kg and/or from about0.1 mg/kg to about 25 mg/kg) of each compound used in the present methodof treatment.

Compositions can be formulated in a unit dosage form, each dosagecontaining from about 1 to about 500 mg of each compound individually orin a single unit dosage form, such as from about 5 to about 300 mg, fromabout 10 to about 100 mg, and/or about 25 mg. The term “unit dosageform” refers to a physically discrete unit suitable as unitary dosagesfor a patient, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical carrier, diluent, orexcipient.

The inert ingredients and manner of formulation of the pharmaceuticalcompositions are conventional. The usual methods of formulation used inpharmaceutical science may be used here. All of the usual types ofcompositions may be used, including tablets, chewable tablets, capsules,solutions, parenteral solutions, intranasal sprays or powders, troches,suppositories, transdermal patches, and suspensions. In general,compositions contain from about 0.5% to about 50% of the compound intotal, depending on the desired doses and the type of composition to beused. The amount of the compound, however, is best defined as the“effective amount”, that is, the amount of the compound which providesthe desired dose to the patient in need of such treatment. The activityof the compounds employed in the compositions and methods disclosedherein are not believed to depend greatly on the nature of thecomposition, and, therefore, the compositions can be chosen andformulated primarily or solely for convenience and economy.

ILLUSTRATIVE EMBODIMENTS

Embodiment 1. A method for killing bacterial persister cells and/ordormant “viable but non-culturable” (VBNC) cells in a bacterialpopulation, the method comprising administering to the bacterialpopulation an effective amount of a compound of Table 1 or Table 4 or asalt or non-salt form thereof.

Embodiment 2. The method of claim 1, wherein the compound is[(5-nitro-3-phenyl-1H-indol-2-yl)methyl]amine having the formula:

or a salt thereof such as [(5-nitro-3-phenyl-1H-indol-2-yl)methyl]aminehydrochloride (NPIMA), having the formula:

Embodiment 3. The method of claim 1, wherein the compound is2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimin-4(3H)-one(BPOET) having the formula:

or a salt thereof.

Embodiment 4. The method of claim 1, wherein the bacterial persistercells and/or VBNC cells are resistant to one or more antibiotics whichdo not comprise any compound of Table 1 or Table 4 or a salt or non-saltform thereof.

Embodiment 5. The method of claim 1, wherein the bacterial persistercells and/or VBNC cells comprise pathogenic Gram-negative bacteria.

Embodiment 6. The method of claim 1, wherein the bacterial persistercells and/or VBNC cells comprise pathogenic Gram-positive bacteria.

Embodiment 7. The method of claim 1, wherein the bacterial persistercells and/or VBNC comprise bacteria selected from Escherichia spp.,Staphylococcus spp., and Pseudomonas spp.

Embodiment 8. The method of claim 1, wherein the bacterial persistercells and/or VBNC comprise E. coli.

Embodiment 9. The method of claim 1, wherein the bacterial persistercells and/or VBNC comprise S. aureus.

Embodiment 10. The method of claim 1, wherein the bacterial persistercells and/or VBNC comprise P. aeruginosa.

Embodiment 11. The method of claim 1, wherein the bacterial persistercells and/or VBNC cells are present in anaerobic conditions.

Embodiment 12. The method of claim 1, wherein the bacterial populationis present in a biofilm.

Embodiment 13. The method of claim 1, wherein the bacterial populationis present in a biofilm and the bacterial persister cells and/or VBNCcells are reduced or eradicated, but the biofilm is not dispersed.

Embodiment 14. The method of claim 1, wherein the bacterial populationis present in an infection in a wound of an individual, and/or whereinthe bacterial population is present in a liquid biological sample orliquid environment (e.g., wherein the bacterial population is present insuspension).

Embodiment 15. The method of claim 1, wherein the population comprisesan infection in an individual, wherein the individual has beenpreviously diagnosed with a bacterial infection and has been treatedwith at least one antibiotic which does not comprise any compound ofTable 1 or Table 4 or a salt or non-salt form thereof.

Embodiment 16. The method of claim 1, further comprising administeringto the bacterial population an antibiotic which does not comprise anycompound of Table 1 or Table 4 or a salt or non-salt form thereof.

Embodiment 17. The method of claim 1, wherein the bacterial persistercells and/or VBNC cells are reduced in the bacterial population.

Embodiment 18. The method of claim 1, wherein the bacterial persistercells and/or VBNC cells are eradicated from the bacterial population.

Embodiment 19. The method of claim 1, wherein a reduction in thebacterial persister cells and/or the VBNC cells in the population occursafter the compound is administered to the population.

Embodiment 20. The method of claim 19, wherein the reduction of thepersister cells and/or the VBNC cells is greater than a reference,wherein the reference comprises a value obtained from reducing persistercells and/or or reducing VBNC cells of the same bacterial species usinga corresponding amount of an antibiotic which does not comprise anycompound of Table 1 or Table 4, such as ciprofloxacin, ampicillin, orgentamicin.

EXAMPLES

The following Examples are illustrative and should not be interpreted tolimit the scope of the claimed subject matter.

Example 1 - Identification of a Potent Indigoid Persister Antimicrobialby Screening Dormant Cells

Reference is made to Song S., et al., “Identification of a PotentIndigoid Persister Antimicrobial by Screening Dormant Cells,”Biotechnology and Bioengineering, Volume 116, Issue 9, pages 2263-2274,04 Jun. 2019, the content of which is incorporated herein by referencein its entirety.

Abstract

The subpopulation of bacterial cells that survive myriad stressconditions (e.g., nutrient deprivation and antimicrobials) by ceasingmetabolism, revive by activating ribosomes. These resuscitated cells canreconstitute infections; hence, it is imperative to discover compoundswhich eradicate persister cells. By screening 10,000 compounds directlyfor persister cell killing, we identified5-nitro-3-phenyl-1Hindol-2-yl-methylamine hydrochloride (NPIMA) as acompound that kills Escherichia coli persister cells more effectivelythan the best indigoid found to date, 5-iodoindole, and better than theDNA-crosslinker cisplatin. In addition, NPIMA eradicated Pseudomonasaeruginosa persister cells in a manner comparable to cisplatin. NPIMAalso eradicated Staphylococcus aureus persister cells but was lesseffective than cisplatin. Critically, NPIMA kills Gram-positive andGram-negative bacteria by damaging membranes and causing lysis asdemonstrated by microscopy and release of extracellular DNA and protein.Furthermore, NPIMA was effective in reducing P. aeruginosa and S. aureuscell numbers in a wound model, and no resistance was found after 1 week.Hence, we identified a potent indigoid that kills persister cells bydamaging their membranes.

1 | INTRODUCTION

Nearly all bacterial cells are stressed (e.g., lack of nutrients andantimicrobials; Kim, Chowdhury, Yamasaki, & Wood, 2018; Song & Wood,2018), so they reduce their metabolism and a subpopulation becomesdormant (Bigger, 1944; Hobby, Meyer, & Chaffee, 1942); this dormantstate is known as persistence. Beyond being prevalent in theenvironment, persistence is relevant in medicine since these cellslikely reconstitute recurring infections (Van den Bergh, Fauvart, &Michiels, 2017). Because traditional antibiotics target growing cellsand are largely ineffective against persister cells that lack themetabolic activity (Defrain, Fauvart, & Michiels, 2018), it is criticalto identify new compounds for killing persister cells to controlinfections.

To target effectively persister cells with new compounds, it is germaneto understand how they form and how they resuscitate. Cells have myriadways to combat stress; for example, they utilize sigma factors like RpoSin Escherichia coli that redirect gene expression upon nutrientdepletion (Wang, Kim, et al., 2011), and most cells in a populationemploy such an active response. However, a subpopulation of cells, as aresult of noisy gene expression or through elegant regulation, becomesdormant (Wood, Song, & Yamasaki, 2019). To reduce metabolism and becomepersistent, cells utilize toxin/antitoxin (TA) systems (Wang & Wood,2011); direct evidence of the importance of specific TA systems inpersistence is that deletion of toxins MqsR (Kim & Wood, 2010;Luidalepp, Jõers, Kaldalu, & Tenson, 2011), TisB (Dörr, Vulić, & Lewis,2010), and YafQ (Harrison et al., 2009) reduces persistence, andproduction of toxins generally increases persistence (Chowdhury, Kwan, &Wood, 2016).

After surviving stress through dormancy, single-cell experimentsdemonstrate E. coli persister cells resuscitate by activating ribosomes(Kim, Yamasaki, Song, Zhang, & Wood, 2018). Resuscitation isheterogeneous as some cells wake immediately and others do not divide orelongate until their ribosome levels are increased (Kim, Yamasaki, etal., 2018). In contrast to ineffective traditional antibiotics, somecompounds have been identified that kill persister cells. For example,two compounds approved by the U.S. Food and Drug Administration foranticancer treatments, mitomycin C and cisplatin, kill Escherichia coli,Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii,and EHEC persister cells by cross-linking their DNA while they aredormant (Chowdhury, Wood, Martínez-Vázquez, García-Contreras, & Wood,2016; Cruz-Muñiz et al., 2016; Kwan, Chowdhury, & Wood, 2015). Asoriginally suggested due to its toxicity (Chowdhury, Wood, et al.,2016), cisplatin has been shown to be effective when applied topicallyfor treating P. aeruginosa infections in a murine keratitis model (Yuanet al., 2018). In addition, Trp/Argcontaining antimicrobial peptideskill persister cells by disrupting the cell structure of the dormantcells (Kwan, Chowdhury, et al., 2015), and ADEP4, an acyldepsipeptideantibiotic, combined with rifampicin, can eradicate S. aureus persistersby causing ClpP protease to degrade proteins nonspecifically (Conlon etal., 2013). By conjugating the traditional antibiotic vancomycin to thecell-penetrating transporter D-octaarginine, S. aureus persisters andbiofilm cells were killed (Antonoplis et al., 2018). In addition, thevitamin A derivatives, retionoids CD437 and CD1530, have been identifiedthat kill S. aureus persisters by disrupting lipid bilayers afterscreening 82,000 small molecules (Kim, Zhu, et al., 2018). Since indolereduces persistence (Hu, Kwan, Osbourne, Benedik, & Wood, 2015; Kwan,Osbourne, Hu, Benedik, & Wood, 2015), indole derivatives, such as5-iodoindole and 4-fluoroindole, have been tested and found to kill E.coli, S. aureus, and EHEC persister cells (Lee, Kim, Gwon, Wood, & Lee,2016), but they are not effective against P. aeruginosa.

In the present study, by screening 10,000 compounds, we identified that5-nitro-3-phenyl-1H-indol-2-yl methylamine hydrochloride (NPIMA) killsE. coli persister cells. In addition, we found the mechanism of killingis via cell lysis. Furthermore, we found that NPIMA is more effectivethan 5-iodoindole with the opportunistic pathogen P. aeruginosa and thatNPIMA eradicates the persister cells of both P. aeruginosa and S.aureus.

2 | MATERIALS AND METHODS 2.1 | Bacteria and Growth Conditions

The bacteria used in this study are E. coli K-12 BW25113 (Baba et al.,2006), S. aureus ATCC29213, and P. aeruginosa PA14 (Liberati et al.,2006). Lysogeny broth (LB; Bertani, 1951) was used at 37° C. forculturing the bacteria. 2-(Aminomethyl)-indole was obtained fromSigma-Aldrich (catalog number 563838), and2-methyl-5-nitro-3-phenyl-1H-indole was obtained from ChemBridge (SanDiego, CA).

2.2 | Persister Cells

E. coli persister cells were prepared following our previous method(Kim, Yamasaki, et al., 2018; Kwan, Valenta, Benedik, & Wood, 2013).Exponentially-growing cells (turbidity of 0.8 at 600 nm) were treatedwith rifampicin (100 µg/ml for 30 min) to stop transcription, washed,and any remaining nonpersister cells were lysed by ampicillin in LB (100µg/ml for 3 hr). Cells were harvested by centrifugation (17,000 g for 1min) and washed with 1× phosphate-buffered saline buffer (PBS, 8 g NaCl,0.2 g KCl, 1.15 g Na₂HPO₄, and 0.2 g KH₂PO₄ per 1,000 ml) twice toremove all possible carbon sources, then resuspended with 1× PBS.Natural E. coli persister cells were generated by treatingstationary-phase cells (turbidity of 6 at 600 nm) with ampicillin (100µg/ml) for 3 hr. P. aeruginosa PA14 persister cells were prepared byincubating to the stationary phase, diluting sixfold, and treating withcarbonyl cyanide m-chlorophenylhydrazone (CCCP, 50 mg/ml stock solutionin dimethly sulfoxide [DMSO]) to stop adenosine triphosphate (ATP)production (200 µg/ml for 3 hr), washed twice with 0.85% NaCl (5,000 gfor 10 min), and any nonpersister cells were killed by ciprofloxacin (5µg/ml) in LB for 3 hr. Following the antibiotic treatment, bacteria werewashed twice with 0.85% NaCl (5,000 g for 10 min).

2.3 | ChemBridge Screen

To identify compounds that kill E. coli persister cells, 10,000compounds of the DIVERset Library from ChemBridge (San Diego, CA) weretested by adding 4 µl of each (in DMSO, final concentration 100 µM) to186 µl of LB in 96-well plates along with 10 µl of persister cells; thepersister cells were added after the ChemBridge chemical since LB wakespersister cells (Kim, Yamasaki, et al., 2018). For the negative control,pure DMSO (final concentration 2 vol%) was used. The degree ofinhibition was determined by the change in turbidity at 600 nm after 24hr. The best 26 compounds were re-tested with the same conditions.

2.4 | Minimum Inhibitory Concentration (MIC)

To determine the MICs of NPIMA and 5-iodoindole for E. coli K-12, S.aureus, and P. aeruginosa PA14, cells were inoculated into LB at varyingconcentrations and grown for 24 hr. The MIC was determined as the lowestconcentration that prevented an increase in growth as evidenced by alack of change of turbidity.

2.5 | Live/Dead Assay

Cell viability after treating with NPIMA was determined using the usingthe LIVE/DEAD BacLight Bacterial Viability Kit (catalog number, L7012;Molecular Probes, Inc., Eugene, OR). The fluorescence signal wasanalyzed via a Zeiss Axioscope.A1 using excitation at 485 nm andemission at 530 nm for green fluorescence and using excitation at 485 nmand emission at 630 nm for red fluorescence.

2.6 | In Vitro Wound Model

Overnight cultures of S. aureus and P. aeruginosa PA14 were diluted inwound-like media (45% Bolton broth, 50% bovine plasma, and 5% lakedhorse blood; Sun, Dowd, Smith, Rhoads, & Wolcott, 2008) to a turbidityof 0.5 at 600 nm in 1 ml. Each culture (1%) was used to inoculate fresh5ml of wound-like media. The combined culture (200 µl/well) was placedinto 96-well plates and incubated at 37° C. with shaking for 24 hr. Thenon-gel liquid was removed, then the gel was washed once with PBS. NPIMA(0.1 and 0.5 mM) and DMSO were then added (200 µl/well) to the plates,which were incubated for another 6 hr with shaking. By triturating, thegel with cells was removed and added to 0.8 ml of PBS, and the cellviability was measured by spreading 100 µl of diluted culture on LBplates.

2.7 | Transmission Electron Microscopy (TEM)

For transmission electron microscopy (TEM), E. coli BW25113 was grown toa turbidity of 0.8 at OD600, contacted with NPIMA at 100 µM for 0.75 hrin PBS, centrifuged at 8000 g, and resuspended in PBS. The samples werefixed with buffer (2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH7.4) and negative stained with 2% uranyl acetate in the dark for 1 hr,then dehydrated. The sectioned specimens were stained again with uranylacetate and lead citrate after dehydration and resin embedded. TEMimages were obtained using a JEOL JEM 1200 EXII instrument.

2.8 | Lysis Assays

Exponentially-growing E. coli BW25113 cells (turbidity 0.8 at 600 nm)were washed twice with 0.85% NaCl, resuspended in 1 ml of 1× PBS, andNPIMA (100 µM) was added for 1 hr for E. coli and P. aeruginosa and 6 hrfor S. aureus with shaking at 250 rpm (0.1% DMSO was used as thenegative control). Cell supernatants were collected after centrifugingat 6,500 g (4° C. for 15 min), and total protein was measured by theBicinchoninic Acid (BCA) protein assay Kit (Prod#23227; Pierce). DNA inthe supernatant (199 µl) was detected by adding 1 µl of Picogreen(P7589; Invitrogen) and incubating for 5 min at room temperature in thedark room. The fluorescence signal was read by a Tecan microplate reader(Infinite M200PRO) with 480 nm excitation and 520 nm emission byutilizing a calibration curved made with plasmid pEX18Ap at 0, 0.004,0.008, 0.016, 0.063, 0.125, 0.25, and 0.5 ng/ µl.

2.9 | Viability and Cytotoxicity Assays

For bacterial viability, cells were washed twice with 0.85% NaCl,resuspended in 1×PBS, and cell counts were determined via the drop assay(Donegan, Matyac, Seidler, & Porteous, 1991). For human cell viability,pre-cultured human cancer HT-29 cells were dispensed in 98 µl in 96-wellplates with approximately 5,000 cells/well. NPIMA was added (2 µl) toproduce concentrations of 5, 10, 100, and 200 µM. For controls, TritonX-100 (positive control) and DMSO (negative solvent control), were used,and the medium was used for background. Plates were incubated in ahumidified incubator (37° C., 5% CO₂) for 24 hr. Cell viability wasdetermined via a cell counting kit (CCK-8 Kit, ab228554; Abcam), andcytotoxicity was determined via the lactate dehydrogenase (LDH) assay(LDH Assay Kit, MK401; Takara).

3 | RESULTS 3.1 | NPIMA Kills E. Coli Persister Cells

To identify compounds capable of killing E. coli persister cells, wecreated a population that consists solely of persister cells and hastheir population increased by 105-fold by pretreatingexponentially-growing cells with rifampicin (100 µg/ml) for 30 min tostop transcription followed by ampicillin treatment (100 µg/ml) for 3 hrto kill any nonpersister cells (Kwan et al., 2013). This method forgenerating persister cells has been evaluated eight ways (Kim, Yamasaki,et al., 2018) and used by us to determine that persister cells wake viaribosome resuscitation (Kim, Yamasaki, et al., 2018) and to show thatthe cells capable of resuscitation in a viable but not culturablepopulation are equivalent to persister cells (Kim, Chowdhury, et al.,2018). In addition, this method has been adopted by at least sixindependent groups (Cui et al., 2018; Grassi et al., 2017; Narayanaswamyet al., 2018; Pu et al., 2019; Sulaiman, Hao, & Lam, 2018;Tkhilaishvili, Lombardi, Klatt, Trampuz, & Di Luca, 2018).

To screen directly for killing persister cells, a high-throughputapproach using 96-well microtiter plates was devised that consisted of(a) washing persister cells formed from exponentially-growing cellsusing rifampicin pretreatment followed by ampicillin treatment, (b)adding 10 µl of the persister cells to 190 µl of LB containing one eachof the 10,000 compounds of the DiverSet library dissolved in dimethylsulfoxide (100 µM final concentration), and (c) monitoring for growthvia change in turbidity for 24 hr. With this approach, we allowed for upto a 140-fold change in turbidity (0.005 could be increased to 0.69).

Table 1 shows the 25 persister cell inhibitors and their structures thatwere identified in the initial screen. Of these 25 persister inhibitors,a second screen was performed under the same conditions as the originalscreen; 8 of these 25 compounds were selected as the most potent (FIG. 1) with NPIMA substantially more effective than the other compounds.Critically, NPIMA was the only compound which reduced the turbidityduring the first and second screen, suggesting NPIMA lyses persistercells. Hence, we focused on this compound.

TABLE 1 Persister cell inhibitors and their structures that wereidentified in the initial screen Name Structure[(5-nitro-3-phenyl-1H-indol-2-yl)methyl]amine hydrochloride (NPIMA)

2-({2-[(4-chlorophenyl)amino]-4-quinazolinyl}amino)ethanol hydrochloride

1-[2-(4-chlorophenoxy)-2-methylpropanoyl]-4-methylpiperazine

N-benzyl-N- {[3 -(4-methoxyphenyl)-1-phenyl- 1H-pyrazol-4-yl]methyl}acetamide

N-(3,4-dichlorophenyl)-N′-(3-fluorophenyl)thiourea

2-({2-[(4-bromophenyl)amino]-4-quinazolinyl}amino)ethanolhydrochloride

N-2-(4-ethoxyphenyl)-2,4-quinazolinediaminehydrochloride

4-{[(5-nitro-2-thienyl)methylene]amino}benzamide

2-[(6-phenyl-2,3,4,9-tetrahydro-1H-carbazol-1-yl)amino]ethanol

2-bromo-4-chlorophenylphenylcarbamate

1-(3,6-dichloro-9H-carbazol-9-yl)-3-(2-methyl-1H-imidazol-1-yl)-2-propanol

N-(3-chloro-4-fluorophenyl)-N′-[2-(difluoromethoxy)phenyl]thiourea

2-(butyryloxy)-1H-benzo[de]isoquinoline-1,3(2H)-dione

N-phenyl-N′-[(1-phenylcyclopentyl)methyl]thiourea

N-[2-(4-fluorophenyl)ethyl]-N′-(4-nitrophenyl)thiourea

2,4-dichloro-5-(5-nitro-2-furyl)benzoic acid

N-(3-chlorophenyl)-N′-[3-(trifluoromethyl)phenyl]thiourea

N-(3-chloro-4-fluorophenyl)-N′-(2-methoxy-4-nitrophenyl)thiourea

4-({[3-chloro-4-fluorophenyl]amino}carbonothioyl)amino-N-ethylbenzenesulfonamide

2-[({2-[(2-chlorobenzyl)oxy]-1-naphthyl}methyl)amino] ethanolhydrochloride

17-(4-bromophenyl)-17-azapentacyclononadeca-2,4,6,9,11,13-hexaene-16,18-dione

N-(3-chloro-4-fluorophenyl)-N′-3-pyridinylthiourea

N-(4-chlorobenzyl)-N′-4-pyridinylthiourea

5-bromo-N-{2-[(4-methylphenyl)thio]ethyl}-2-thiophenesulfonamide

N′-(3,5-dichloro-2-hydroxybenzylidene)-2-oxo-4-phenyl-3-pyrrolidinecarbohydrazide

3.2 | NPIMA Kills E. Coli Exponential Cells

Since NPIMA was identified as killing persister cells, we tested whetherit is effective on both persister cells and exponential cells. The MICfor NPIMA with E. coli was determined to be 100 µM (15 µg/ml) (Table 2);hence, we tested it at 100 µM (1 MIC) and found NPIMA eradicatesexponentially-growing E. coli within 3 hr (FIG. 2 a ). Furthermore,NPIMA (100 µM) also eradicated E. coli persisters in 6 hr (FIG. 3 a ).Critically, persister cells generated with only ampicillin treatment(“natural persisters”) were killed in an identical manner (FIG. 9 ),which confirms our rifampicin-treatment persister model. Therefore,NPIMA is kills both persister and exponentially-growing E. coli.

TABLE 2 MICs (mM) for 5-iodoindole, NPIMA, and cisplatin Strain5-lodoindole NPIMA Cisplatin Escherichia coli BW251113 2 0.1 0.3Staphylococcus aureus 2 0.1 1 Pseudomonas aeruginosa PA14 2 0.25 0.15Note: Values for cisplatin are from (Chowdhury, Wood, et al., 2016).Abbreviations: MIC, minimum inhibitory concentration; NPIMA,5-nitro-3-phneyl-1H-indol-2-yl-methylamine hydrochloride.

3.3 | NPIMA Damages the Cell Membrane and Causes Cell Lysis

To initially explore how NPIMA kills cells, we treatedexponentially-growing E. coli BW25113 cells with 100 µM NPIMA andstained with the LIVE/DEAD kit. Remarkably, we found that cells treatedwith 100 µM NPIMA lysed as evidenced by the extracellular DNA seensurrounding cells that was stained by both Syto9 and propidium iodide ofthe LIVE/DEAD kit (FIG. 4 ); note there were no dead cells seen with thesolvent control (DMSO). Corroborating the cells lysis seen with the DNAdyes, treatment of exponentially-growing E. coli with 100 µM NPIMA for 1hr led to both a 10 ± 2 increase in total cell protein in supernatantsas well as a 3.8 ± 0.8 increase in DNA in the supernatants compared tothe addition of DMSO alone (FIG. 5 ). To investigate further the celllysis caused by NPIMA, we used transmission electron microscopy andfound clear cell envelope damage (FIG. 6 ). Together, these five linesof evidence show NPIMA lyses E. coli cells.

3.4 | NPIMA Is More Effective With E. Coli Persisters Than 5-Iodoindole

Since 5-iodoindole is the most effective indigo derivative for killingE. coli persister cells (kills 99.993% of persisters at 1 mM; Lee etal., 2016), we compared the effectiveness of this compound to NPIMA.Using both compounds at 100 µM, as indicated above, NPIMA eradicatedboth exponential (FIG. 2 a ), persister cells (FIG. 3 a ) of E. coliwhereas 5-iodoindole was much less effective. Corroborating theseresults, we found the MIC for 5-iodoindole to be 2 mM (Table 2)

3.5 | NPIMA Has Broad Activity

We also tested whether NPIMA was effective at killing P. aeruginosa andS. aureus persister cells. At 100 µM, NPIMA eradicatedexponentially-growing P. aeruginosa PA14 cells in 6 hr (FIG. 2 b ) aswell as eradicated P. aeruginosa persister cells in 3 hr (FIG. 3 b ).Similarly, NPIMA eradicated S. aureus cells at 200 µM in 3 hr (FIG. 2 c). In contrast, 5- iodoindole was ineffective with both P. aeruginosa at100 µM and S. aureus at 200 µM. Corroborating these results, the MIC for5-iodoindole for P. aeruginosa was 2 mM versus 0.25 mM for NPIMA, andthe MIC for 5-iodoindole for S. aureus was 2 mM versus 0.1 mM for NPIMA(Table 2).Therefore, NPIMA is highly effective with P. aeruginosa and S.aureus.

3.6 | NPIMA Lyses S. Aureus and P. Aeruginosa

We also investigated the mechanism by which NPIMA kills S. aureus and P.aeruginosa persister cells. Using the LIVE/DEAD staining Kit, we foundafter 1 hr, 100 µM NPIMA killed 27% for the S. aureus cells (FIG. 4 c ).Critically, we also saw evidence of S. aureus cell lysis in the form ofhazy staining with Syto9 only around cells with NPIMA. Hence, we checkedfor the presence of extracellular DNA and protein as evidence of lysisand found 2.9 ± 0.1-fold total protein and 1.82 ± 0.05-fold DNA releaseddue to 100 µM NPIMA treatment after 6 hr compared with the addition ofDMSO alone (FIG. 5 ).

Similar to S. aureus, 100 µM NPIMA also lysed P. aeruginosa as indicatedby LIVE/DEAD staining that shows distinct extracellular DNA aftertreating for 1 hr for both Syto9 and propidium iodide (FIG. 4 b );however, unlike with S. aureus, all (100%) of the P. aeruginosa cellswere killed in 1 hr. Therefore, we checked for the presence ofextracellular DNA and protein as evidence of lysis and found a 1.19 ±0.09-fold increase in DNA and a 4.0 ± 0.4-fold total protein releaseddue to 100 µM NPIMA treatment after 1 hr compared to the addition ofDMSO alone (FIG. 5 ). Hence, as with E. coli, NPIMA lyses bothGram-positive S. aureus and Gram-negative P. aeruginosa.

3.7 | Wound Model

To test NPIMA against the pathogen P. aeruginosa and S. aureus in arealistic infection model, we chose the in vitro Lubbock chronic woundpathogenic biofilm model (Sun et al., 2008), since both pathogens arefrequently found together in wounds (DeLeon et al., 2014) and this modelmimics the conditions of polyclonal infections. We found NPIMA (0.5 mM)reduced the total viable number of cells of S. aureus and P. aeruginosain the wound model 10-fold in 6 hr compared to the DMSO solvent control(FIG. 10 ).

3.8 | NPIMA Is More Effective for E. Coli but Less Effective for P.Aeruginosa and S. Aureus Than the DNA Crosslinker Cisplatin

To gauge its effectiveness, we compared NPIMA to cisplatin, which hasbeen shown to be effective for killing P. aeruginosa (Chowdhury, Wood,et al., 2016; Yuan et al., 2018), with each compound used at one MIC(Table 2). As shown in FIG. 7 a , cisplatin eradicated P. aeruginosacells in 1 hr whereas NPIMA was less effective. For E. coli (FIG. 7 b ),NPIMA was more effective than cisplatin, but for S. aureus (FIG. 7 c ),NPIMA was less effective than cisplatin.

3.9 | Resistance

To test if E. coli could obtain resistance easily to NPIMA, cells werepropagated daily in LB with 0.25 MIC of NPIMA (25 µM) for 7 days. After7 days, the sequentially-propagated E. coli cells were contacted with LBwith 1 MIC of NPIMA (100 µM) and incubated overnight to allow anyputative resistant cells to grow and increase the turbidity. Critically,all of the E. coli cells were killed. Hence, resistance to NPIMA doesnot occur readily.

3.10 | Structure Activity Relationships

To discern insights about the importance of the three substituents onthe indole ring of NPIMA, we tested the importance of both the nitro andphenyl groups by assaying the killing of 2-(aminomethyl)-indole with E.coli and found 2-(aminomethyl)-indole (100 µM) is unable to kill E. coli(FIG. 8 ). In addition, 2-methyl-5-nitro-3-phenyl-1H-indole was used toascertain the importance of the amine group, and we found2-methyl-5-nitro-3-phenyl-1H-indole (100 µM) is unable to kill E. coli(FIG. 8 ). Hence, all three substituents are important for NPIMAactivity.

3.11 | NPIMA Cytotoxicity

To determine the cytotoxicity of NPIMA, we performed both an LDH assayand CCK-8 assay with human cells. NPIMA was not toxic at 5 and 10 µM butshowed toxicity in both tests at concentrations of 50 µM and higher(FIG. 11 ).

4 | DISCUSSION

Previously, we demonstrated through two independent approaches that cellsignaling through indole decreases persistence (Hu et al., 2015; Kwan,Osbourne, et al., 2015). Specifically, by producing the RNase toxin YafQof the E. coli YafQ/DinJ TA system, we found YafQ cleaves the mRNA oftryptophanase, which produces indole from tryptophan; hence, there isless indole production and a dramatic increase in persistence (Hu etal., 2015). Additionally, we showed that producing the phosphodiesteraseDosP reduces cAMP concentrations which in turn reduces tryptophanase andindole production which leads to a dramatic increase in persistence(Kwan, Osbourne, et al., 2015).

Also, direct addition of both indole and halogenated indoles reducespersistence (Lee et al., 2016); hence, indole signaling reducespersister cell formation. For compounds to kill persister cells bycorrupting cytosolic functions, they must be able to enter the cytosolof the dormant cell through passive diffusion, like the DNA-crosslinkersmitomycin C (Kwan, Chowdhury, et al., 2015; Wood, 2016) and cisplatin(Chowdhury, Wood, et al., 2016) or they can attack the outside of thecell by damaging the membrane, like retinoids (Kim, Zhu, et al., 2018).Critically, we found here that NPIMA, a substituted indole, reducespersistence, not by changing indole signaling and altering tryptophanaseactivity (Hu et al., 2015; Kwan, Osbourne, et al., 2015), but by killingcells through lysis from membrane damage (FIGS. 4-6 ). This mode ofkilling was found to be general for both Gram-negative and Gram-positivebacteria since we found NPIMA was effective with E. coli, P. aeruginosa,and S. aureus. NPIMA is probably less effective in the complete lysis ofGram-positive strains (FIG. 4 ), which results in the release ofcellular protein and DNA, due to the protective cell wall ofGram-positive strains that Gram-negative strains lack. Although there isless lysis of Gram-positive S. aureus, NPIMA kills S. aureus as well asE. coli (FIG. 2 ); hence, complete lysis of Gram-positive strains mustnot be necessary for NPIMA to cause cell death. Furthermore, comparingGram-negative strains, actively growing P. aeruginosa was lysed (FIGS. 4and 5 ) and killed (FIG. 2 ) less effectively than actively-growing E.coli, most likely due to the innate resistance of P. aeruginosa due toits active efflux (Chen et al., 2010). However, NPIMA was able to killthe most recalcitrant of cells, persister cells, of all three bacteriasince it eradicated the generated persister cells of E. coli and P.aeruginosa equally well (FIG. 3 ) as well as eradicated the completepopulation of S. aureus cells, which includes persisters (FIG. 2 c ).Note that due to its cytotoxicity at 50 µM to human cells, NPIMA wouldhave to be used in combination with other antimicrobials or less toxicderivatives need to be found.

Previously, indole (Chimerel, Field, Piñero-Fernandez, Keyser, &Summers, 2012) and the indole derivative 1-geranylindole (Yang et al.,2017)[5-fluoro-[(E)-1-(3,7-dimethylocta-2,6-dien-1-yl)]-3-(piperidin-1-ylmethyl)-1H-indole]have been shown to disrupt the cell membrane. In addition,1-geranylindole killed nongrowing Mycobacterium bovis; however, unlikeNPIMA, 1-geranylindole has no effect on E. coli (Yang et al., 2017).Therefore, we have discovered a potent substituted indole that iseffective in killing a wide-range of bacteria.

REFERENCES

Antonoplis, A., Zang, X., Huttner, M. A., Chong, K. K. L., Lee, Y. B.,Co, J. Y.,... Cegelski, L. (2018). A dual-functionantibiotic-transporter conjugate exhibits superior activity insterilizing MRSA biofilms and killing persister cells. Journal of theAmerican Chemical Society, 140, 16140-16151.

Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., ...Mori, H. (2006). Construction of Escherichia coli K-12 in-frame,single-gene knockout mutants: The Keio collection. Molecular SystemsBiology, 2, 2006.0008.

Bertani, G. (1951). Studies on lysogenesis .1. The mode of phageliberation by lysogenic Escherichia coli. Journal of Bacteriology, 62,293-300.

Bigger, J. W. (1944). Treatment of staphylococcal infections withpenicillin-By intermittent sterilisation. Lancet, 2, 497-500.

Chen, H., Yi, C., Zhang, J., Zhang,W., Ge, Z., Yang, C.-G., & He, C.(2010). Structural insight into the oxidation-sensing mechanism of theantibiotic resistance of regulator MexR. EMBO Reports, 11, 685-690.

Chimerel, C., Field, C. M., Piñero-Fernandez, S., Keyser, U. F., &Summers, D. K. (2012). Indole prevents Escherichia coli cell division bymodulating membrane potential. Biochimica et BiophysicaActa-Biomembranes, 1818, 1590-1594.

Chowdhury, N., Kwan, B. W., & Wood, T. K. (2016). Persistence increasesin the absence of the alarmone guanosine tetraphosphate by reducing cellgrowth. Scientific Reports, 6, 20519.

Chowdhury, N., Wood, T. L., Martínez-Vázquez, M., Garcia-Contreras, R.,& Wood, T. K. (2016). DNA-crosslinker cisplatin eradicates bacterialpersister cells. Biotechnology and Bioengineering, 113, 1984-1992.

Conlon, B. P., Nakayasu, E. S., Fleck, L. E., LaFleur, M. D., Isabella,V. M., Coleman, K., ... Lewis, K. (2013). Activated ClpP killspersisters and eradicates a chronic biofilm infection. Nature, 503,365-370.

Cruz-Muñiz, M. Y., López-Jacome, L. E., Hernández-Durán, M.,Franco-Cendejas, R., Licona-Limón, P., Ramos-Balderas, J. L., ...Garcia-Contreras, R. (2016). Repurposing the anticancer drug mitomycin Cfor the treatment of persistent Acinetobacter baumannii infections.International Journal of Antimicrobial Agents, 49, 88-92.

Cui, P., Niu, H., Shi, W., Zhang, S., Zhang, W., & Zhang, Y. (2018).Identification of genes involved in bacteriostatic antibiotic-inducedpersister formation. Frontiers in Microbiology, 9, 413.

Defrain, V., Fauvart, M., & Michiels, J. (2018). Fighting bacterialpersistence: Current and emerging anti-persister strategies andtherapeutics. Drug Resistance Updates, 38, 12-26.

DeLeon, S., Clinton, A., Fowler, H., Everett, J., Horswill, A. R., &Rumbaugh, K. P. (2014). Synergistic Interactions of Pseudomonasaeruginosa and Staphylococcus aureus in an in vitro wound model.Infection and Immunity, 82, 4718-4728.

Donegan, K., Matyac, C., Seidler, R., & Porteous, A. (1991). Evaluationof methods for sampling, recovery, and enumeration of bacteria appliedto the phylloplane. Applied and Environmental Microbiology, 57, 51-56.

Dörr, T., Vulić, M., & Lewis, K. (2010). Ciprofloxacin causes persisterformation by inducing the TisB toxin in Escherichia coli. PLOS Biology,8, e1000317.

Grassi, L., Di Luca, M., Maisetta, G., Rinaldi, A. C., Esin, S.,Trampuz, A., & Batoni, G. (2017). Generation of persister cells ofPseudomonas aeruginosa and Staphylococcus aureus by chemical treatmentand evaluation of their susceptibility to membrane-targeting agents.Frontier in Microbiology, 8, 1917.

Harrison, J. J., Wade,W. D., Akierman, S., Vacchi-Suzzi, C., Stremick,C. A., Turner, R. J., & Ceri, H. (2009). The chromosomal toxin gene yafQis a determinant of multidrug tolerance for Escherichia coli growing ina biofilm. Antimicrobial Agents and Chemotherapy, 53, 2253-2258.

Hobby, G. L., Meyer, K., & Chaffee, E. (1942). Observations on themechanism of action of penicillin. Proceedings of the Society forExperimental Biology and Medicine, 50, 281-285.

Hu, Y., Kwan, B. W., Osbourne, D. O., Benedik, M. J., & Wood, T. K.(2015). Toxin YafQ increases persister cell formation by reducing indolesignalling. Environmental Microbiology, 17, 1275-1285.

Kim, J.-S., Chowdhury, N., Yamasaki, R., & Wood, T. K. (2018). Viablebut non-culturable and persistence describe the same bacterial stressstate. Environmental Microbiology, 20, 2038-2048.

Kim, J.-S., Yamasaki, R., Song, S., Zhang, W., & Wood, T. K. (2018).Single cell observations show persister cells wake based on ribosomecontent. Environmental Microbiology, 20, 2085-2098.

Kim, W., Zhu, W., Hendricks, G. L., VanTyne, D., Steele, A. D., Keohane,C. E., ... Mylonakis, E. (2018). A new class of synthetic retinoidantibiotics effective against bacterial persisters. Nature, 556,103-107.

Kim, Y., & Wood, T. K. (2010). Toxins Hha and CspD and small RNAregulator Hfq are involved in persister cell formation through MqsR inEscherichia coli. Biochemical Biophysical Ressearch Communications, 391,209-213.

Kwan, B. W., Chowdhury, N., & Wood, T. K. (2015). Combatting bacterialinfections by killing persister cells with mitomycin C. EnvironmentalMicrobiology, 17, 4406-4414.

Kwan, B. W., Osbourne, D. O., Hu, Y., Benedik, M. J., & Wood, T. K.(2015). Phosphodiesterase DosP increases persistence by reducing cAMPwhich reduces the signal indole. Biotechnology and Bioengineering, 112,588-600.

Kwan, B. W., Valenta, J. A., Benedik, M. J., & Wood, T. K. (2013).Arrested protein synthesis increases persister-like cell formation.Antimicrobial Agents and Chemotherapy, 57, 1468-1473.

Lee, J.-H., Kim, Y.-G., Gwon, G., Wood, T. K., & Lee, J. (2016).Halogenated indoles eradicate bacterial persister cells and biofilms.AMB Express, 6, 123.

Liberati, N. T., Urbach, J. M., Miyata, S., Lee, D. G., Drenkard, E.,Wu, G., ... Ausubel, F. M. (2006). An ordered, nonredundant library ofPseudomonas aeruginosa strain PA14 transposon insertion mutants.Proceeding of the National Academy of Sciences of United States ofAmerica, 103, 2833-2838.

Luidalepp, H., Jõers, A., Kaldalu, N., & Tenson, T. (2011). Age ofinoculum strongly influences persister frequency and can mask effects ofmutations implicated in altered persistence. Journal of Bacteriology,193, 3598-3605.

Narayanaswamy, V. P., Keagy, L. L., Duris, K., Wiesmann, W., Loughran,A. J., Townsend, S. M., & Baker, S. (2018). Novel glycopolymereradicates antibiotic-and CCCP-induced persister cells in Pseudomonasaeruginosa. Frontier in Microbiology, 9, 1724.

Pu, Y., Li, Y., Jin, X., Tian, T., Ma, Q., Zhao, Z., ... Bai, F. (2019).ATP dependent dynamic protein aggregation regulates bacterial dormancydepth critical for antibiotic tolerance. Molecular Cell, 73, 143-156.

Song, S., & Wood, T. K. (2018). Post-segregational killing and phageinhibition are not mediated by cell death through toxin/antitoxinsystems. Frontier in Microbiology, 9, 814.

Sulaiman, J. E., Hao, C., & Lam, H. (2018). Specific enrichment andproteomics analysis of escherichia escherichia coli coli persisters fromrifampin pretreatment. Journal of Proteome Research, 17, 3984-3996.

Sun, Y., Dowd, S. E., Smith, E., Rhoads, D. D., & Wolcott, R. D. (2008).In vitro multispecies Lubbock chronic wound biofilm model. Wound Repairand Regeneration, 16, 805-813.

Tkhilaishvili, T., Lombardi, L., Klatt, A.-B., Trampuz, A., & Di Luca,M. (2018). Bacteriophage Sb-1 enhances antibiotic activity againstbiofilm, degrades exopolysaccharide matrix and targets persisters ofStaphylococcus aureus. International Journal of Antimicrobial Agents,52, 842-853.

Vanden Bergh, B., Fauvart, M., & Michiels, J. (2017). Formation,physiology, ecology, evolution and clinical importance of bacterialpersisters. FEMS Microbiology Reviews, 41, 219-251.

Wang, X., Kim, Y., Hong, S. H., Ma, Q., Brown, B. L., Pu, M., ... Wood,T. K. (2011). Antitoxin MqsA helps mediate the bacterial general stressresponse. Nature Chemical Biology, 7, 359-366.

Wang, X., & Wood, T. K. (2011). Toxin-antitoxin systems influencebiofilm and persister cell formation and the general stress response.Applied and Environmental Microbiology, 77, 5577-5583.

Wood, T. K. (2016). Combatting bacterial persister cells. Biotechnologyand Bioengineering, 113, 476-483.

Wood, T. K., Song, S., & Yamasaki, R. (2019). Ribosome dependence ofpersister cell formation and resuscitation. Journal of Microbiology, 57,213-219.

Yang, T., Moreira, W., Nyantakyi, S. A., Chen, H., Aziz, Db, Go, M.-L.,& Dick, T. (2017). Amphiphilic indole derivatives as antimycobacterialagents: Structure-activity relationships and membrane targetingproperties. Journal of Medicinal Chemistry, 60,2745-2763.

Yuan, M., Chua, S. L., Liu, Y., Drautz-Moses, D. I., Yam, J. K. H.,Aung, T. T., ... Nielsen, T. E. (2018). Repurposing the anticancer drugcisplatin with the aim of developing novel Pseudomonas aeruginosainfection control agents. Beilstein Journal of Organic Chemistry, 14,3059-3069.

Example 2 - Persister Cells Resuscitate via Ribosome Modification by 23SrRNA Pseudouridine Synthase RluD

Reference is made to Song S. and Wood TK., “Persister Cells Resuscitatevia Ribosome Modification by 23S rRNA Pseudouridine Synthase RluD,”bioRxiv, doi: https://doi.org/10.1101/678425, available on-line Jun. 21,2019; now published in Environmental Microbiology doi:10.1111/1462-2920.14828, Volume 22, Issue 3, Pages 850-857, March 2020,first published 13 Oct. 2019.

ABSTRACT

Upon a wide range of stress conditions (e.g., nutrient, antibiotic,oxidative), a subpopulation of bacterial cells known as persisterssurvive by halting metabolism. These cells resuscitate rapidly toreconstitute infections once the stress is removed and nutrients areprovided. However, how these dormant cells resuscitate is not understoodwell but involves reactivating ribosomes. By screening 10,000 compoundsdirectly for stimulating Escherichia coli persister cell resuscitation,we identified that2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[l]benzothieno[2,3-d]pyrimidin-4(3H)-one(BPOET) stimulates resuscitation. Critically, by screening 4,267 E. coliproteins, we determined that BPOET activates hibernating ribosomes via23S rRNA pseudouridine synthase RluD, which increases ribosome activity.Corroborating the increased waking with RluD, production of RluDincreased the number of active ribosomes in persister cells. Also,inactivating the small RNA RybB which represses rluD led to fasterpersister resuscitation. Hence, persister cells resuscitate viaactivation of RluD.

INTRODUCTION

Upon myriad stresses such as antibiotic stress, a sub-population ofbacterial cells becomes dormant and multi-stress tolerant (1, 2); thesecells are known as persisters. The persister phenotype is not due togenetic change, since upon re-growth, persisters cells behave the sameas the original culture. Persistence is relevant in the environmentsince almost all cells face starvation (3) and relevant in medicinesince recurring infections may be the result of regrowth of persistercells (4). The persister sub-population should be distinguished fromslow-growing cells such as those in the stationary-phase or thosegenerated by nutrient shifts (5); these slow-growing cells may bedistinguished from persisters since the whole population of slow-growingcells are tolerant to antimicrobials whereas the non-growing persisterpopulation is a small sub-population (less than 1%) (6). Thisdistinction is critical since tolerant cells utilize alternate sigmafactors like RpoS in Escherichia coli to redirect gene expression as anactive response against stress (7), whereas persisters cease respondingand become dormant (5, 8).

To treat persister cell infections, it is important to understand howthey form and how they resuscitate. The prevailing view for theirformation (6) is that to reduce metabolism, cells activate toxins oftoxin/antitoxin (TA) systems (9). The best genetic evidence for this isthat deletion of toxins MqsR (10, 11). TisB (12), and YafQ (13)decreases persistence. Moreover, production of non-TAs toxins alsoincreases persistence (14). However, since nutrient deprivation alsoresults in persistence (15), the sub-population of cells may becomedormant simply by running out of food. In addition, we have proposed amodel whereby the alarmone ppGpp (synthesized as a result of myriadstress conditions), directly creates persister cells via ribosomedimerization, without the need of TA systems (16). Regardless of themechanism, persistence appears to be an elegantly-regulated response toan unfavorable environment (17).

In regard to resuscitating persister cells, little has been determinedabout the mechanism. It has been suggested that persister cellsresuscitate by inactivating toxins such as TacT acetyltransferase viapeptidyl-tRNA hydrolase Pth (18), but this has not been demonstrated. Itis established that persister cells revive in response primarily toenvironmental signals, such as fresh nutrients (rather thanstochastically) (19). In addition, persisters revive in an heterogeneousmanner, by activating ribosomes; cells increase their ribosome contentuntil a threshold is reached, then they begin to elongate or divide(19). For resuscitation, the persisters sense nutrients by chemotaxisand phosphotransferase membrane proteins, reduce cAMP levels to rescuestalled ribosomes, unhybridize 100S ribosomes via HflX, and undergochemotaxis toward fresh nutrients (20).

In the present study, to discern additional insights into how ribosomesare active as persister cells resuscitate, we converted the complete E.coli population into persister cells so that we could screen for thefirst time compounds that enhance persister resuscitation. From a 10,000compound library, we identified that2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one(BPOET) stimulates persister cell waking. Critically, we determined thatthe mechanism by which BPOET resuscitates persisters is via activationof the 23S rRNA pseudouridine synthase RluD, which is important forribosome activity. Hence, BPOET stimulates persister resuscitation byactivating ribosomes via RluD.

MATERIALS AND METHODS

Bacteria and growth conditions. E. coli K-12 and its isogenic mutants(Table 3) were grown routinely in lysogeny broth (21) (22) at 37° C.BPOET was obtained from ChemBridge (San Diego, CA).

TABLE 3 E. coli bacterial strains and plasmids used in this study. KmRand CmR indicate kanamycin and chloramphenicol resistance, respectively.Strains and Plasmids Features Source BW25113 Wild type (39) BW25113ΔrluD ΔrluD Km^(R) (39) MG1655 Wild type (40) MG1655 ΔrybB ΔrybB Km^(R)(41) MG1655□ASV rrnbP1::GFP[ASV] (26) Plasmids pCA24N Cm^(R); lacI^(q)(25) pCA24N_rluD Cm^(R); lacI^(q), P_(T5-Inc)::rluD⁺ (25)

Persister cells. E. coli persister cells were generated (19, 23) bytreating exponentially-growing cells (turbidity of 0.8 at 600 nm) withrifampicin (100 µg/mL) for 30 min to stop transcription, centrifuging,and adding LB with ampicillin (100 µg/mL) for 3 h to lyse anynon-persister cells. Cells pellets were washed twice with 0.85% NaClthen re-suspended in 0.85% NaCl.

ChemBridge screen to identify resuscitation compounds. To identifycompounds that resuscitate E. coli persister cells, the DIVERset Libraryfrom ChemBridge (San Diego, CA) containing 10,000 druglike compoundswith high pharmacophore diversity was evaluated by adding 4 µL of eachcompound (final concentration 100 µM, dissolved in DMSO) to 186 µL of LBin 96 well plates and then adding 10 µL of persister cells. The negativecontrol was 2 vol% DMSO. Resuscitation was calculated as the change inturbidity at 600 nm. The compounds that were identified initially werere-tested in M9 minimal medium with 5X alanine (24).

Pooled ASKA screen to identify resuscitation proteins. To identifyproteins responsible for resuscitation, all 4,267 ASKA clones (GFP-)(25) were combined, grown to a turbidity of 2 at 600 nm in LB medium,and their plasmids isolated using a plasmid DNA Mini Kit I (OMEGABio-Tek, Inc., Norcross, GA, USA). The pooled ASKA plasmids (1 µLcontaining 30 ng DNA) were electroporated into 50 µL of E. coli BW25113competent cells, 1 mL LB medium was added, and the cells were grown to aturbidity of 0.5 at 600 nm. Chloramphenicol was added (30 µg/mL) to theculture to maintain the plasmids, and the cells were incubated at 250rpm to a turbidity of 0.8. Rifampicin followed by ampicillin was addedto make persister cells, then the persister cells were washed twice with1x PBS buffer, contacted with 100 µM BPOET for 2 h in M9 medium thatlacked a carbons source, and plated on LB (Cm) agar plates. Fastercolony appearance indicated faster persister resuscitation. Plasmidswere isolated from the colonies and sequenced using primer pCA24N_F:GCCCTTTCGTCTTCACCTCG.

Single-cell persister resuscitation. As described previously (19), 5 µLof cell populations consisting of 100% persister cells were added to1.5% agarose gel pads containing either M9 medium with glucose (0.4 wt%)or alanine (5X) as a carbon source (24), and resuscitation was monitoredat 37° C. via a light microscope (Zeiss Axio Scope.A1, bl_ph channel at1000 ms exposure).

Active 70S ribosome assay. The GFP signal of resuscitating persisters ofE. coli K-12 MG1655-ASVGFP (26) with RluD was monitored using afluorescence microscope (Zeiss Axioscope.A1, bl_ph channel at 1,000 msexposure and GFP channel at 10,000 ms exposure). E. coli K-12MG1655-ASVGFP produces an unstable variant of GFP (half-life less than 1h) under the control of the 16S rRNA ribosomal promoter rrnbP1 (26).

RESULTS & DISCUSSION

BPOET resuscitates E. coli persister cells. To identify compounds thatresuscitate E. coli persister cells, we first increased by 10⁵-fold thepersister cell population by pre-treating with rifampicin to ceasemetabolism by stopping transcription followed by ampicillin treatment tokill any remaining non-persister cells (23). In this way, nearly 100% ofbacterial cell population was converted into persister cells. Hence, wewere able to both screen for compounds that more rapidly resuscitatepersister cells as well as confirm our hypotheses via single-cellmicroscopy. The persister cells generated in this way have been (i)confirmed eight ways (19), (ii) used to determine that persister cellswake via ribosome activation (19) and chemotaxis (20), (iii) used toshow that the cells capable of resuscitation in a viable but notculturable population are equivalent to persister cells (15). (iv) usedto identify compounds that kill persister cells (27), and (v) used toshow that the alarmone ppGpp directly creates persister cells bystimulating ribosome dimerization (16). In addition, our method togenerate a high population of persister cells has been utilized by atleast six independent groups (28-33).

Using 96-well plates, the persister cells (10 µL) were added to 190 µLof LB containing one each of the 10,000 compounds of the DiverSetlibrary dissolved in dimethyl sulfoxide (100 µM final concentration),and growth was monitored via the change in turbidity for up to 48 h.Starting at a turbidity of 0.05, a 140-fold increase in growth waspossible (maximum final turbidity of 0.69). Table 4 shows the 27compounds that were identified that stimulated persister cellresuscitation relative to the negative control of dimethyl sulfoxide.

TABLE 4 Compounds that resuscitate persister cells and their structuresthat were identified in the initial screen. Name Structure2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one (BPOET)

methyl-5-[(dimethylamino)carbonyl]-4-methyl-2-({[(1-methyl-1H-pyrazol-3-yl)amino]carbonothioyl}amino)-3-thiophenecarboxylate

N-[2-(3,4-dimethoxyphenyl)ethyl]-N′-[1-(pentafluorobenzyl)-1H-pyrazol-3-yl]thiourea

4-chloro-N-(6,7-dimethoxy-4-oxo-1,4-dihydro-2-quinazolinyl)benzamide

(4-methoxyphenyl)(phenyl)methanone

N-(3-acetylphenyl)-4,5-dimethyl-2-furamide

6-(4-iodophenyl)-2-methylimidazo[2,1-b][1,3]thiazole

N-{[(4-bromophenyl)amino]carbonothioyl}-2,2-dimethylpropanamide

1-(2,4-dichlorobenzoyl)-2,3-dihydro-1h-imidazo[1,2-a]benzimidazole

3-(3-chlorophenyl)-5,5-dimethyl-4-methylene-1,3-oxazolidin-2-one

2-methyl-4-(4-(methylthio)phenyl)-5-oxoN-phenyl-1,4,5,6,7,8-hexahydro-3-quinolinecarboxamide

4-(isopropoxycarbonyl)benzyl2-pyrazinecarboxylate

N-[4-(2-oxo-1-pyrrolidinyl)phenyl]-1H-1,2,4-triazole-3-carboxamide

4-chloro-N-(4-oxo-1,4-dihydro-2-quinazolinyl)benzamide

3-hydroxy-5-(4-propoxyphenyl)-1-(3-pyridinylmethyl)-4-(2-thienylcarbonyl)-1,5-dihydro-2H-pyrrol-2-one

4-(3,4-dimethoxyphenyl)-2-hydrazino-6-phenylpyrimidine

N′-[1-(3,4-dimethoxyphenyl)ethylidene]-3-phenyl-1H-pyrazole-5-carbohydrazide

3-[4-(4-chlorophenyl)-1-piperazinyl]-1-(4-iodophenyl)-2,5-pyrrolidinedione

N-(3-oxo-1,3-dihydro-2-benzofuran-5-yl)-1H-1,2,4-triazole-3-carboxamide

3-[(5-methyl-2-furoyl)amino]benzoicacid

N-(4-{[(2,4-dimethoxyphenyl)amino]sulfonyl}phenyl)-3-[(4-methylphenyl)thio]propanamide

N~2~-(3-fluorophenyl)-N~2~-(methylsulfonyl)-N~1~-[2-(1-pyrrolidinylcarbonyl)phenyl]glycinamide

N-(5-chloro-2-methoxyphenyl)-N′-(1-ethyl-3,5-dimethyl-1H-pyrazol-4-yl)thiourea

1-[(4-methylphenyl)sulfonyl]-N-1,3-thiazol-2-ylprolinamide

2,5-dichloro-N-(2-furylmethyl)benzamide

3-{[(2-methoxyphenyl)amino]methyl}-5-[4-(methylthio)benzylidene]-1,3-thiazolidine-2,4-dione

5-(4-propoxybenzyl)-1H-tetrazole

Upon confirming the results of these initial hits in minimal alaninemedium, we found BPOET (100 µM) was most effective and increasedpersister cell waking by 44-fold in 96-well plates based on theincreases in turbidity as well as found that BPOET increases the wakingof single persister cells by 4-fold (FIG. 12 , Table 5). Hence, wefocused on this compound.

Table 5. Single persister cell resuscitation. Single-cell persisterresuscitation as determined using light microscopy (Zeiss Axio Scope.A1)using agarose gel pads. Microscope images are shown in FIG. 13 . Thefold-change in resuscitation is relative to BW25113 with DMSO forBW25113 with BPOET, relative to BW25113 for the ΔrluD, relative toBW25113/pCA24N for the strain producing RluD from pCA24N in BW25113, andrelative to MG1655 for ΔrybB. M9 glucose (0.4%) agarose gel pads wereused for all the strains except BW25113 with BPOET where M9 alanine (5X)agarose gel pads including 100 µM of BPOET or DMSO were used. Theresults are the combined observations from two independent experimentsafter 6 h for the BW25113 with BPOET and DMSO, after 4 h for BW25113 andits deletion mutants, and after 6 h for cells harboring pCA24N and itsderivatives as well as for MG1655, and ΔrybB. Standard deviations areshown, and each strain was visualized at 14 positions.

Total cells Waking cells % waking Fold-change BW25113 on DMSO 215 ± 4611 ± 4 5 ± 2 1 BW25113 on BPOET 213 ± 49 38 ± 18 20 ± 13 4 ± 3 BW25113150± 46 24 ± 5 16 ± 1 1 ΔrluD 327 ± 16 5 ± 0 1.5 ± 0.1 -10 ± 1 pCA24N233 ± 155 5 ± 4 1.8 ± 0.3 1 pCA24N-rluD 210 ± 33 43 ± 6 20 ± 6 11 ± 4MG1655 310 ± 103 8.5 ± 5 2.6 ± 0.7 1 ΔrybB 208 ± 10 45 ± 0 22 ± 1 8 ± 2

Table 6._Active 70S ribosomes in single persister cells forMG1655-ASV/pCA24N-rulD (“pCA24N-rluD”) vs. MG1655-ASV/pCA24N (“pCA24N”).Single-cell persister resuscitation as determined using light microscopy(Zeiss Axio Scope.A1) using agarose gel pads with 0.4% glucose.Microscope images are shown in FIG. 12B. The fold-change inresuscitation is relative to MG1655-ASV/pCA24N forM1655-ASV/pCA24N-rluD.

pCA24N-rluD pCA24N Total cells 140 ± 66 112 ± 4 High intensity cells 120± 64 25 ± 3 Waking % 85 ± 6 22 ± 2 Fold-change 3.8 ± 0.4 1

BPOET resuscitates E. coli persister cells by modifying ribosomes. Todetermine how BPOET resuscitates persister cells, we pooled the 4,267ASKA clones in which each E. coli protein is produced from plasmidpCA24N, produced persister cells carrying these plasmids, contacted with100 µM BPOET, plated the cells, and chose the largest colonies thatformed on LB plates. Our rationale was that any pathway stimulated byBPOET would be even more active if the number of rate-limiting proteinsin that pathway were increased, and cells that wake first would formcolonies faster.

Using this approach, we identified five proteins whose productionincreased resuscitation: RluD, YjiK, SrlR, Smf, and YeeZ. These proteinsare related to contacting with BPOET since addition of the diluent DMSOalone and sequencing larger colonies did not identify these fiveproteins but instead identified TmcA, a tRNA^(Met) cytidineacetyltransferase, which is a general factor required for translationthat likely led to larger colony sizes with the diluent. Of the proteinsrelated to BPOET, only RluD (23S rRNA pseudouridine synthase) and SrlR(represses the gut operon for glucitol metabolism) have beencharacterized; we focused on RluD because it is related to ribosomes,and we have shown inactivating ribosomes causes persistence (23) andactivating ribosomes resuscitates persister cells (16, 19, 20). RluD isinvolved in the synthesis and assembly of 70S ribosomes as well as theirfunction based on its post-transcriptional modification of 23S rRNA toform three pseudouridine (5-ribosyl-uracil) nucelosides at positions1911, 1915, and 1917 (34). In pseudouridine, uracil is attached via acarbon-carbon bond to the sugar base rather than through acarbon-nitrogen bond. The 23S rRNA pseudouridines increase the stabilityof the tertiary structure of 23S rRNA and are located in a stem loopstructure that is involved in peptidyltransferase and interacts withmRNA, tRNA, 16S rRNA, and ribosome release factor. Hence, RluD isresponsible for efficient ribosome function (34).

RluD enhances persister cell resuscitation. To explore further the roleof RluD and persister resuscitation, we utilized single cell studiessince persister cells are heterogeneous (19) and wake with differentfrequencies (which would be missed if we monitored planktonicpopulations). We found that deleting rulD reduces the frequency ofsingle-cell persister resuscitation dramatically (11-fold) compared tothe isogenic wild-type strain on minimal glucose agarose gel pads (FIG.12A, Table 5, FIG. 13 ). In addition, no colonies were found on M9glucose agar plates after inactivating RluD (FIG. 14 ), confirming thatpersister cells are severely challenged in resuscitation without RluD.Corroborating these two results with the rluD mutant, production of RluDincreased the frequency of waking by 11-fold on glucose medium (FIG.12A, Table 5, FIG. 13 ) and increased waking on rich medium (results notshown). In addition, the rluD deletion has no effect on persister cellformation (FIG. 14 ). Therefore, RluD stimulates persister cellresuscitation but does not affect persister formation.

RluD increases active ribosomes for resuscitation. Using a GFP reporterthat indicates the number of 70S ribosomes in individual persister cells(19), we found that producing RluD before making persister cells makes85 ± 6% of the cells have high ribosome fractions compared to notproducing RluD (FIG. 12B). The GFP reporter indicates transcription ofrrsB (16S rRNA), gltT (tRNA-glu), rrlB (23S rRNA) and rrfB (5S rRNA);hence, it indicates production of the three major rRNA building blocks.Although this is not a direct observation of 70S ribosomes, this methodis a suitable proxy for the number of ribosomes based on measurement ofrRNA concentrations and has been used frequently (19, 35-37), and wehave verified its use by isolating ribosomes and comparing GFPfluorescence (19). Hence, the increased persister cell resuscitationwith RluD is directly due to the increase in active (70S) ribosomes ofpersister cells.

RybB antagonizes persister cell resuscitation. Since the small RNA RybBrepresses RluD (38), we investigated its impact on persisterresuscitation. As expected, we found that deletion of rybB increases thefrequency of persister cell waking by 8-fold (FIG. 12A, Table 5, FIG. 13).

In summary, the results presented here demonstrate that ribosomes may beactivated for specific cell cycles such as recovery from dormancy.Specifically, by screening for compounds for the first time that enhancepersister cell resuscitation, we have (i) determined that ribosomes aremodified by RluD as cells resuscitate and resume ribosome activity, (ii)identified a novel compound, BPOET, that activates persister cells, and(iii) linked small RNAs to persistence. Hence, these results extend ourunderstanding of how persister cells are activated which has afar-reaching impact in that all bacteria cope with nutrient stress andbecome dormant.

REFERENCES

1. Hobby GL, Meyer K, & Chaffee E Observations on the mechanism ofaction of penicillin. P Soc Exp Biol Med 50:281-285 (1942).

2. Bigger JW Treatment of staphylococcal infections with penicillin - Byintermittent sterilisation. Lancet 2:497-500 (1944).

3. Song S & Wood TK Post-segregational Killing and Phage Inhibition AreNot Mediated by Cell Death Through Toxin/Antitoxin Systems. FrontMicrobiol 9:814 (2018).

4. Van den Bergh B, Fauvart M, & Michiels J Formation, physiology,ecology, evolution and clinical importance of bacterial persisters. FEMSMIcrobiol. Rev. 41:219-251 (2017).

5. Kim J-S & Wood TK Tolerant, Growing Cells from Nutrient Shifts AreNot Persister Cells. mBio 8:e00354-00317 (2017).

6. Ronneau S & Helaine S Clarifying the Link between Toxin-AntitoxinModules and Bacterial Persistence. J. Mol. Biol. (2019).

7. Wang X, et al. Antitoxin MqsA helps mediate the bacterial generalstress response. Nature Chem. Biol. 7:359-366 (2011).

8. Kim J-S & Wood TK Persistent Persister Misperceptions. Front.Microbiol. 7:2134 (2016).

9. Wang X & Wood TK Toxin-antitoxin systems influence biofilm andpersister cell formation and the general stress response. Appl. Environ.Microbiol. 77:5577-5583 (2011).

10. Kim Y & Wood TK Toxins Hha and CspD and small RNA regulator Hfq areinvolved in persister cell formation through MqsR in Escherichia coli.Biochem. Biophys. Res. Commun. 391:209-213 (2010).

11. Luidalepp H, Jõers A, Kaldalu N, & Tenson T Age of Inoculum StronglyInfluences Persister Frequency and Can Mask Effects of MutationsImplicated in Altered Persistence. J. Bacteriol. 193:3598-3605 (2011).

12. Dörr T, Vulic M, & Lewis K Ciprofloxacin causes persister formationby inducing the TisB toxin in Escherichia coli. PLoS Biol. 8:e1000317(2010).

13. Harrison JJ, et al. The chromosomal toxin gene yafQ is a determinantof multidrug tolerance for Escherichia coli growing in a biofilm.Antimicrob. Agents Chemother. 53:2253-2258 (2009).

14. Chowdhury N, Kwan BW, & Wood TK Persistence Increases in the Absenceof the Alarmone Guanosine Tetraphosphate by Reducing Cell Growth.Scientific Reports 6:20519 (2016).

15. Kim J-S, Chowdhury N, Yamasaki R, & Wood TK Viable ButNon-Culturable and Persistence Describe the Same Bacterial Stress State.Environ Microbiol 20:2038-2048 (2018).

16. Song S & Wood TK ppGpp Ribosome Dimerization Model for BacterialPersister Formation and Resuscitation. bioRxiv: 663658 (2019).

17. Wood TK, Song S, & Yamasaki R Ribosome dependence of persister cellformation and resuscitation. J. Microbiol. 57:DOI10.1007/s12275-12019-18629-12272 (2019).

18. Cheverton Angela M, et al. A Salmonella Toxin Promotes PersisterFormation through Acetylation of tRNA. Mol. Cell 63:86-96 (2016).

19. Kim J-S, Yamasaki R, Song S, Zhang W, & Wood TK Single CellObservations Show Persister Cells Wake Based on Ribosome Content.Environ. Microbiol. 20:2085-2098 (2018).

20. Yamasaki R, Song S, Benedik MJ, & Wood TK Persister CellsResuscitate Using Membrane Sensors that Activate Chemotaxis, Lower cAMPLevels, and Revive Ribosomes. bioRxiv doi 10.1101/486985:486985 (2019).

21. Aizenman E, Engelberg-Kulka H, & Glaser G An Escherichia colichromosomal “addiction module” regulated by guanosine3′,5′-bispyrophosphate: a model for programmed bacterial cell death.Proc Natl Acad Sci USA 93:6059-6063 (1996).

22. Bertani G Studies on Lysogenesis .1. The Mode of Phage Liberation byLysogenic Escherichia-Coli. J. Bacteriol. 62:293-300 (1951).

23. Kwan BW, Valenta JA, Benedik MJ, & Wood TK Arrested proteinsynthesis increases persister-like cell formation. Antimicrob. AgentsChemother. 57:1468-1473 (2013).

24. Rodriguez RL & Tait RC (1983) Recombinant DNA Techniques: AnIntroduction (Benjamin/Cummings Publishing, Menlo Park, CA).

25. Kitagawa M, et al. Complete set of ORF clones of Escherichia coliASKA library (a complete set of E. coli K-12 ORF archive): uniqueresources for biological research. DNA Res 12:291-299 (2005).

26. Shah D, et al. Persisters: a distinct physiological state of E.coli. BMC Microbiol 6:53 (2006).

27. Song S, Gong T, Yamasaki R, Kim J-S, & Wood TK Identification of aPotent Indigoid Persister Antimicrobial by Screening Dormant Cells.Biotechnol. Bioengr. https://doi.org/10.1002/bit.27078 (2019).

28. Cui P, et al. Identification of Genes Involved in BacteriostaticAntibiotic-Induced Persister Formation. Front Microbiol 9:413 (2018).

29. Grassi L, et al. Generation of Persister Cells of Pseudomonasaeruginosa and Staphylococcus aureus by Chemical Treatment andEvaluation of Their Susceptibility to Membrane-Targeting Agents. FrontMicrobiol 8:1917 (2017).

30. Narayanaswamy VP, et al. Novel Glycopolymer EradicatesAntibiotic-and CCCP-Induced Persister Cells in Pseudomonas aeruginosa.Front Microbiol 9:1724 (2018).

31. Pu Y, et al. ATP-Dependent Dynamic Protein Aggregation RegulatesBacterial Dormancy Depth Critical for Antibiotic Tolerance. MolecularCell 73:1-14 (2019).

32. Sulaiman JE, Hao C, & Lam H Specific Enrichment and ProteomicsAnalysis of Escherichia coli Persisters from Rifampin Pretreatment. JProteome Res 17:3984-3996 (2018).

33. Tkhilaishvili T, Lombardi L, Klatt A-B, Trampuz A, & Di Luca MBacteriophage Sb-1 enhances antibiotic activity against biofilm,degrades exopolysaccharide matrix and targets persisters ofStaphylococcus aureus. Int J Antimicrob Agents 52:842-853 (2018).

34. Gutgsell NS, Deutsher MP, & Ofengand J The pseudouridine synthaseRluD is required for normal ribosome assembly and function inEscherichia coli. RNA 11:1141-1152 (2005).

35. Burger K, et al. Chemotherapeutic Drugs Inhibit Ribosome Biogenesisat Various Levels. J Biol Chem 285:12416-12425 (2010).

36. Lu T, Stroot PG, & Oerther DB Reverse Transcription of 16S rRNA ToMonitor Ribosome-Synthesizing Bacterial Populations in the Environment.Appl Environ Microb 75:4589-4598 (2009).

37. Piques M, et al. Ribosome and transcript copy numbers, polysomeoccupancy and enzyme dynamics in Arabidopsis. Mol Syst Biol 5:314(2009).

38. Gogol EB, Rhodius VA, Papenfort K, Vogel J, & Gross CA Small RNAsendow a transcriptional activator with essential repressor functions forsingle-tier control of a global stress regulon. Proc. Natl. Acad. Sci.U.S.A.:201109379 (2011).

39. Baba T, et al. Construction of Escherichia coli K-12 in-frame,single-gene knockout mutants: the Keio collection. Mol. Syst. Biol.2:2006 0008 (2006).

40. Guyer MS, Reed RR, Steitz JA, & Low KB Identification of asex-factor-affinity site in E. coli as gamma delta. Cold Spring Harborsymposia on quantitative biology 45 Pt 1:135-140 (1981).

41. Hobbs EC, Astarita JL, & Storz G Small RNAs and Small ProteinsInvolved in Resistance to Cell Envelope Stress and Acid Shock inEscherichia coli: Analysis of a Bar-Coded Mutant Collection. J.Bacteriol. 192:59-67 (2010).

In the foregoing description, it will be readily apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations which is not specifically disclosed herein.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention. Thus, it should be understood that although the presentinvention has been illustrated by specific embodiments and optionalfeatures, modification and/or variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

1-20. (canceled)
 21. A method for killing bacterial persister cellsand/or dormant “viable but non-culturable” (VBNC) cells in a bacterialpopulation, the method comprising administering to the bacterialpopulation an effective amount of a compound of[2-({2-[4-chlorophenyl)amino]4-quinazolinyl}amino) ethanolhydrochloride,1-[2-(4-chlorophenoxy)-2-methylpropanoyl]-4-methylpiperazine,N-benzyl-N-{[3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl]methyl}acetamide,N-(3,4-dichlorophenyl)-N′-(3-fluorophenyl)thiourea,2-({2-[(4-bromophenyl)amino]-4-quinazolinyl}amino)ethanol hydrochloride,N-2-(4-ethoxyphenyl)-2,4-quinazolinediamine hydrochloride, 4-{[(5-nitro-2-thienyl)methylene]amino}benzamide,2-[(6-phenyl-2,3,4,9-tetrahydro-1H-carbazol-1-yl)amino]ethanol,2-bromo-4-chlorophenyl phenylcarbamate,1-(3,6-dichloro-9H-carbazol-9-yl)-3-(2-methyl-1H-imidazol-1-yl)-2-propanol,N-(3-chloro-4-fluorophenyl)-N′-[2-(difluoromethoxy)phenyl]thiourea,2-(butyryloxy)-1H-benzo[de]isoquinoline-1,3(2H)-dione,N-phenyl-N′-[(1-phenylcyclopentyl)methyl]thiourea,N-[2-(4-fluorophenyl)ethyl]-N′-(4-nitrophenyl(thiourea,2,4-dichloro-5-(5-nitro-2-furyl)benzoic acid,N-(3-chlorophenyl)-N′-[3-(trifluoromethyl)phenyl]thiourea,N-(3-chloro-4-fluorophenyl)-N′-(2-methoxy-4-nitrophenyl)thiourea, 4-({[3-chloro-4-fluorophenyl]amino}carbonothioyl)amino-N-ethylbenzenesulfonamide,2-[({2-[(2-chlorobenzyl)oxy]-1-naphthyl }methyl)amino]ethanolhydrochloride,17-(4-bromophenyl)-17-azapentacyclononadeca-2,4,6,9,11,13-hexaene-16,18-dione,N-(3-chloro-4-fluorophenyl)-N′-3-pyridinylthiourea,N-(4-chlorobenzyl)-N′-4-pyridinylthiourea,5-bromo-N-{2-[(4-methylphenyl)thio]ethyl}-2-thiophenesulfonamide,N′-(3,5-dichloro-2-hydroxybenzylidene)-2-oxo-4-phenyl-3-pyrrolidinecarbohydrazide,2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one,methyl5-[(dimethylamino)carbonyl]-4-methyl-2-({[(1-methyl-1H-pyrazol-3-yl)amino]carbonothioyl(amino)-3-thiophenecarboxylate,N-[2-(3,4-dimethoxyphenyl)ethyl]N′-[1-(pentafluorobenzyl)-1H-pyrazol-3-yl]thiourea,4-chloro-N-(6,7-dimethoxy-4-oxo-1,4-dihydro-2-quinazolinyl)benzamide,(4-methoxyphenyl)(phenyl) methanone,N-(3-acetylphenyl)-4,5-dimethyl-2-furamide,6-(4-iodophenyl)-2-methylimidazo[2,1-b][1,3]thiazole,N-{[(4-bromophenyl)amino]carbonothioyl}-2,2-dimethylpropanamide,1-(2,4-dichlorobenzoyl)-2,3-dihydro-1H-i-imidazo[1,2-a]benzimidazole,3-(3-chlorophenyl)-5,5-dimethyl-4-methylene-1,3-oxazolidin-2-one,2-methyl-4-[4-(methylthio)phenyl]-5-oxo-N-phenyl-1,4,5,6,7,8-hexahydro-3-quinolinecarboxamide,4-(isopropoxycarbonyl)benzyl 2-pyrazinecarboxylate,N-[4-(2-oxo-1-pyrrolidinyl)phenyl]-1H-1,2,4-triazole-3-carboxamide,4-chloro-N-(4-oxo-1,4-dihydro-2-quinazolinyl)benzamide,3-hydroxy-5-(4-propoxyphenyl)-1-(3-pyridinylmethyl)-4-(2-thienylcarbonyl)-1,5-dihydro-2H-pyrrol-2-one,4-(3,4-dimethoxyphenyl)-2-hydrazino-6-phenylpyrimidine,N′-[1-(3,4-dimethoxyphenyl)ethylidene]-3-phenyl-1H-pyrazole-5-carbohydrazide,3-[4-(4-chlorophenyl)-1-piperazinyl]-1-(4-iodophenyl)-2,5-pyrrolidinedione,N-(3-oxo-1,3-dihydro-2-benzofuran-5-yl)-1H-1,2,4-triazole-3-carboxamide,3-[(5-methyl-2-furoyl)amino]benzoicacid,N-(4-{[(2,4-dimethoxyphenyl)amino]sulfonyl}phenyl)-3-[(4-methylphenyl)thio]propenamide,N~2~-(3-fluorophenyl)N~2~-(methylsulfonyl)N~1~-[2-(1-pyrrolidinylcarbonyl)phenyl]glycinamide,N-(5-chloro-2-methoxyphenyl)-N′-(1-ethyl-3,5-dimethyl-1H-pyrazol-4-yl)thiourea,1-[(4-methylphenyl)sulfonyl]N-1,3-thiazol-2-ylprolinamide,2,5-dichloro-N-(2-furylmethyl)benzamide, and3-{[(2-methoxyphenyl)amino]methyl}-5-[4-(methylthio)benzylidene]-1,3-thiazolidine-2,4-dione,5-(4-propoxybenzyl)-1H-tetrazole, or a salt or non-salt form thereof.22. The method of claim 21, wherein the compound is2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimin-4(3H)-one(BPOET)having the formula:

or a salt thereof.
 23. The method of claim 21, wherein the bacterialpersister cells and/or VBNC cells are resistant to one or moreantibiotics which do not comprise any compound of[5-nitro-3-phenyl-1H-indol-2-yl)methyl]amine hydrochloride,[2-({2-[4-chlorophenyl)amino]4-quinazolinyl}amino) ethanolhydrochloride,1-[2-(4-chlorophenoxy)-2-methylpropanoyl]-4-methylpiperazine,N-benzyl-N-{[3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl]methyl}acetamide,N-(3,4-dichlorophenyl)-N′-(3-fluorophenyl)thiourea,2-({2-[(4-bromophenyl)amino]-4-quinazolinyl}amino)ethanol hydrochloride,N-2-(4-ethoxyphenyl)-2,4-quinazolinediamine hydrochloride, 4-{[(5-nitro-2-thienyl)methylene]amino}benzamide,2-[(6-phenyl-2,3,4,9-tetrahydro-1H-carbazol-1-yl)amino]ethanol,2-bromo-4-chlorophenyl phenylcarbamate,1-(3,6-dichloro-9H-carbazol-9-yl)-3-(2-methyl-1H-imidazol-1-yl)-2-propanol,N-(3-chloro-4-fluorophenyl)-N′-[2-(difluoromethoxy)phenyl]thiourea,2-(butyryloxy)-1H-benzo[de]isoquinoline-1,3(2H)-dione,N-phenyl-N′-[(1-phenylcyclopentyl)methyl]thiourea,N-[2-(4-fluorophenyl)ethyl]-N′-(4-nitrophenyl)thiourea,2,4-dichloro-5-(5-nitro-2-furyl)benzoic acid,N-(3-chlorophenyl)-N′-[3-(trifluoromethyl)phenyl]thiourea,N-(3-chloro-4-fluorophenyl)-N′-(2-methoxy-4-nitrophenyl)thiourea, 4-({[3-chloro-4-fluorophenyl]amino}carbonothioyl)amino-N-ethylbenzenesulfonamide,2-[({2-[(2-chlorobenzyl)oxy]-1-naphthyl }methyl)amino]ethanolhydrochloride,17-(4-bromophenyl)-17-azapentacyclononadeca-2,4,6,9,11,13-hexaene-16,18-dione,N-(3-chloro-4-fluorophenyl)-N′-3-pyridinylthiourea,N-(4-chlorobenzyl)-N′-4-pyridinylthiourea,5-bromo-N-{2-[(4-methylphenyl)thio]ethyl}-2-thiophenesulfonamide,N′-(3,5-dichloro-2-hydroxybenzylidene)-2-oxo-4-phenyl-3-pyrrolidinecarbohydrazide,2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one,methyl5-[(dimethylamino)carbonyl]-4-methyl-2-({[(1-methyl-1H-pyrazol-3-yl)amino]carbonothioyl}amino)-3-thiophenecarboxylate,N-[2-(3,4-dimethoxyphenyl)ethyl]N′-[1-(pentafluorobenzyl)-1H-pyrazol-3-yl]thiourea,4-chloro-N-(6,7-dimethoxy-4-oxo-1,4-dihydro-2-quinazolinyl)benzamide,(4-methoxyphenyl)(phenyl) methanone,N-(3-acetylphenyl)-4,5-dimethyl-2-furamide,6-(4-iodophenyl)-2-methylimidazo[2,1-b][1,3]thiazole,N-{[(4-bromophenyl)amino]carbonothioyl}-2,2-dimethylpropanamide,]-(2,4-dichlorobenzoyl)-2,3-dihydro-1H-imidazo[1,2-a]benzimidazole,3-(3-chlorophenyl)-5,5-dimethyl-4-methylene-1,3-oxazolidin-2-one,2-methyl-4-[4-(methylthio)phenyl]-5-oxo-N-phenyl-1,4,5,6,7,8-hexahydro-3-quinolinecarboxamide,4-(isopropoxycarbonyl)benzyl 2-pyrazinecarboxylate,N-[4-(2-oxo-1-pyrrolidinyl)phenyl]-1H-1,2,4-triazole-3-carboxamide,4-chloro-N-(4-oxo-1,4-dihydro-2-quinazolinyl)benzamide,3-hydroxy-5-(4-propoxyphenyl)-1-(3-pyridinylmethyl)-4-(2-thienylcarbonyl)-1,5-dihydro-2H-pyrrol-2-one,4-(3,4-dimethoxyphenyl)-2-hydrazino-6-phenylpyrimidine,N′-[1-(3,4-dimethoxyphenyl)ethylidene]-3-phenyl-1H-pyrazole-5-carbohydrazide,3-[4-(4-chlorophenyl)-1-piperazinyl]-1-(4-iodophenyl)-2,5-pyrrolidinedione,N-(3-oxo-1,3-dihydro-2-benzofuran-5-yl)-1H-1,2,4-triazole-3-carboxamide,3-[(5-methyl-2-furoyl)amino]benzoicacid,N-(4-{[(2,4-dimethoxyphenyl)amino]sulfonyl}phenyl)-3-[(4-methylphenyl)thio]propenamide,N~2~-(3-fluorophenyl)N~2~-(methylsulfonyl)N~1~-[2-(1-pyrrolidinylcarbonyl)phenyl]glycinamide,N-(5-chloro-2-methoxyphenyl)-N′-(1-ethyl-3,5-dimethyl-1H-pyrazol-4-yl)thiourea,1-[(4-methylphenyl)sulfonyl]N-1,3-thiazol-2-ylprolinamide,2,5-dichloro-N-(2-furylmethyl)benzamide, and3-{[(2-methoxyphenyl)amino]methyl}-5-[4-(methylthio)benzylidene]-1,3-thiazolidine-2,4-dione,5-(4-propoxybenzyl)-1H-tetrazole, or a salt or non-salt form thereof.24. The method of claim 21, wherein the bacterial persister cells and/orVBNC cells comprise pathogenic Gram-negative bacteria.
 25. The method ofclaim 21, wherein the bacterial persister cells and/or VBNC cellscomprise pathogenic Gram-positive bacteria.
 26. The method of claim 21,wherein the bacterial persister cells and/or VBNC comprise bacteriaselected from Escherichia spp., Staphylococcus spp., and Pseudomonasspp.
 27. The method of claim 21, wherein the bacterial persister cellsand/or VBNC comprise bacteria Escherichia spp.
 28. The method of claim21, wherein the bacterial persister cells and/or VBNC comprise E. coli.29. The method of claim 21, wherein the bacterial persister cells and/orVBNC comprise S. aureus.
 30. The method of claim 21, wherein thebacterial persister cells and/or VBNC comprise P. aeruginosa.
 31. Themethod of claim 21, wherein the bacterial persister cells and/or VBNCcells are present in anaerobic conditions.
 32. The method of claim 21,wherein the bacterial population is present in a biofilm.
 33. The methodof claim 21, wherein the bacterial population is present in a biofilmand the bacterial persister cells and/or VBNC cells are reduced oreradicated, but the biofilm is not dispersed.
 34. The method of claim21, wherein the bacterial population is present in an infection in awound of an individual, and/or wherein the bacterial population ispresent in a liquid biological sample or liquid environment (e.g.,wherein the bacterial population is present in suspension).
 35. Themethod of claim 21, wherein the population comprises an infection in anindividual, wherein the individual has been previously diagnosed with abacterial infection and has been treated with at least one antibioticwhich does not comprise any compound of[5-nitro-3-phenyl-1H-indol-2-yl)methyl]amine hydrochloride,[2-({2-[4-chlorophenyl)amino]4-quinazolinyl}amino) ethanolhydrochloride,1-[2-(4-chlorophenoxy)-2-methylpropanoyl]-4-methylpiperazine,N-benzyl-N-{[3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl]methyl}acetamide,N-(3,4-dichlorophenyl)-N′-(3-fluorophenyl)thiourea,2-({2-[(4-bromophenyl)amino]-4-quinazolinyl}amino)ethanol hydrochloride,N-2-(4-ethoxyphenyl)-2,4-quinazolinediamine hydrochloride,4-{[(5-nitro-2-thienyl)methylene]amino}benzamide,2-[(6-phenyl-2,3,4,9-tetrahydro-1H-carbazol-1-yl)amino]ethanol,2-bromo-4-chlorophenyl phenylcarbamate,1-(3,6-dichloro-9H-carbazol-9-yl)-3-(2-methyl-1H-imidazol-1-yl)-2-propanol,N-(3-chloro-4-fluorophenyl)-N′-[2-(difluoromethoxy)phenyl]thiourea,2-(butyryloxy)-1H-benzo[de]isoquinoline-1,3(2H)-dione,N-phenyl-N′-[(1-phenylcyclopentyl)methyl]thiourea,N-[2-(4-fluorophenyl)ethyl]-N′-(4-nitrophenyl(thiourea,2,4-dichloro-5-(5-nitro-2-furyl)benzoic acid,N-(3-chlorophenyl)-N′-[3-(trifluoromethyl)phenyl]thiourea,N-(3-chloro-4-fluorophenyl)-N′-(2-methoxy-4-nitrophenyl)thiourea,4-({[3-chloro-4-fluorophenyl]amino}carbonothioyl)amino-N-ethylbenzenesulfonamide,2-[({2-[(2-chlorobenzyl)oxy]-1-naphthyl }methyl)amino]ethanolhydrochloride,17-(4-bromophenyl)-17-azapentacyclononadeca-2,4,6,9,11,13-hexaene-16,18-dione,N-(3-chloro-4-fluorophenyl)-N′-3-pyridinylthiourea,N-(4-chlorobenzyl)-N′-4-pyridinylthiourea,5-bromo-N-{2-[(4-methylphenyl)thio]ethyl}-2-thiophenesulfonamide,N′-(3,5-dichloro-2-hydroxybenzylidene)-2-oxo-4-phenyl-3-pyrrolidinecarbohydrazide,2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one,methyl5-[(dimethylamino)carbonyl]-4-methyl-2-({[(1-methyl-1H-pyrazol-3-yl)amino]carbonothioyl}amino)-3-thiophenecarboxylate,N-[2-(3,4-dimethoxyphenyl)ethyl]N′-[1-(pentafluorobenzyl)-1H-pyrazol-3-yl]thiourea,4-chloro-N-(6,7-dimethoxy-4-oxo-1,4-dihydro-2-quinazolinyl)benzamide,(4-methoxyphenyl)(phenyl) methanone,N-(3-acetylphenyl)-4,5-dimethyl-2-furamide,6-(4-iodophenyl)-2-methylimidazo[2,1-b][1,3]thiazole,N-{[(4-bromophenyl)amino]carbonothioyl}-2,2-dimethylpropanamide,1-(2,4-dichlorobenzoyl)-2,3)-dihydro-1H-imidazo[1,2-a]benzimidazole,3-(3-chlorophenyl)-5,5-dimethyl-4-methylene-1,3-oxazolidin-2-one,2-methyl-4-[4-(methylthio)phenyl]-5-oxo-N-phenyl-1,4,5,6,7,8-hexahydro-3-quinolinecarboxamide,4-(isopropoxycarbonyl)benzyl 2-pyrazinecarboxylate,N-[4-(2-oxo-1-pyrrolidinyl)phenyl]-1H-1,2,4-triazole-3-carboxamide,4-chloro-N-(4-oxo-1,4-dihydro-2-quinazolinyl)benzamide,3-hydroxy-5-(4-propoxyphenyl)-1-(3-pyridinylmethyl)-4-(2-thienylcarbonyl)-1,5-dihydro-2H-pyrrol-2-one,4-(3,4-dimethoxyphenyl)-2-hydrazino-6-phenylpyrimidine,N′-[1-(3,4-dimethoxyphenyl)ethylidene]-3-phenyl-1H-pyrazole-5-carbohydrazide,3-[4-(4-chlorophenyl)-1-piperazinyl]-1-(4-iodophenyl)-2,5-pyrrolidinedione,N-(3-oxo-1,3-dihydro-2-benzofuran-5-yl)-1H-1,2,4-triazole-3-carboxamide,3-[(5-methyl-2-furoyl)amino]benzoicacid,N-(4-{[(2,4-dimethoxyphenyl)amino]sulfonyl}phenyl)-3-[(4-methylphenyl)thio]propenamide,N∼2∼(3-fluorophenyl)N∼2∼-(methylsulfonyl)N∼1∼-[2-(1-pyrrolidinylcarbonyl)phenyl]glycinamide,N-(5-chloro-2-methoxyphenyl)-N′-(1-ethyl-3,5-dimethyl-1H-pyrazol-4-yl)thiourea,1-[(4-methylphenyl)sulfonyl]N-1,3-thiazol-2-ylprolinamide,2,5-dichloro-N-(2-furylmethyl)benzamide, and 3-{[(2-methoxyphenyl)amino]methyl}-5-[4-(methylthia)benzylidene]-1,3-thiazolidine-2,4-dione,5-(4-propoxybenzyl)-1H-tetrazole or a salt or non-salt form thereof. 36.The method of claim 21, further comprising administering to thebacterial population an antibiotic which does not comprise any compoundof [5-nitro-3-phenyl-1H-indol-2-yl)methyl]amine hydrochloride,[2-({2-[4-chlorophenyl)amino]4-quinazolinyl}amino) ethanolhydrochloride,1-[2-(4-chlorophenoxy)-2-methylpropanoyl]-4-methylpiperazine,N-benzyl-N-{[3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl]methyl}acetamide,N-(3,4-dichlorophenyl)-N′-(3-fluorophenyl)thiourea,2-({2-[(4-bromophenyl)amino]-4-quinazolinyl}amino)ethanol hydrochloride,N-2-(4-ethoxyphenyl)-2,4-quinazolinediamine hydrochloride, 4- {[(5-nitro-2-thienyl)methylene]amino}benzamide,2-[(6-phenyl-2,3,4,9-tetrahydro-1H-carbazol-1-yl)amino]ethanol,2-bromo-4-chlorophenyl phenylcarbamate,1-(3,6-dichloro-9H-carbazol-9-yl)-3-(2-methyl-1H-imidazol-1-yl)-2-propanol,N(3-chloro-4-fluorophenyl)-N′-[2-(difluoromethoxy)phenyl]thiourea,2-(butyryloxy)-1H-benzo[de]isoquinoline-1,3(2H)-dione,N-phenyl-N′-[(1-phenylcyclopentyl)methyl]thiourea,N-[2-(4-fluorophenyl)ethyl]-N′-(4-nitrophenyl)thiourea,2,4-dichloro-5-(5-nitro-2-furyl)benzoic acid,N(3-chlorophenyl)-N′-[3-(trifluoromethyl)phenyl]thiourea,N-(3-chloro-4-fluorophenyl)-N′-(2-methoxy-4-nitrophenyl)thiourea,4-({[3-chloro-4-fluorophenyl]amino}carbonothioyl)amino-Nethylbenzenesulfonamide,2-[({2-[(2-chlorobenzyl)oxy]-1-naphthyl}methyl)amino]ethanolhydrochloride,17-(4-bromophenyl)-17-azapentacyclononadeca-2,4,6,9,11,13-hexaene-16,18-dione,N-(3-chloro-4-fluorophenyl)-N′-3-pyridinylthiourea,N-(4-chlorobenzyl)-N′-4-pyridinylthiourea,5-bromo-N-{2-[(4-methylphenyl)thio]ethyl}-2-thiophenesulfonamide,N′-(3,5-dichloro-2-hydroxybenzylidene)-2-oxo-4-phenyl-3-pyrrolidinecarbohydrazide,2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one,methyl5-[(dimethylamino)carbonyl]-4-methyl-2-({[(1-methyl-1H-pyrazol-3-yl)amino]carbonothioyl}amino)-3-thiophenecarboxylate,N-[2-(3,4-dimethoxyphenyl)ethyl]N′-[1-(pentafluorobenzyl)-1H-pyrazol-3-yl]thiourea,4-chloro-N-(6,7-dimethoxy-4-oxo-1,4-dihydro-2-quinazolinyl)benzamide,(4-methoxyphenyl)(phenyl) methanone,N-(3-acetylphenyl)-4,5-dimethyl-2-furamide,6-(4-iodophenyl′)-2-methylimidazo[2.1-b][1,3]thiazole,N-{[(4-bromophenyl)amino]carbonothioyl}-2,2-dimethylpropanamide,1-(2,4-dichlorobenzoyl)-2,3-dihydro-1H-imidazo[1,2-a]benzimidazole,3-(3-chlorophenyl)-5,5-dimethyl-4-methylene-1,3-oxazolidin-2-one,2-methyl-4-[4-(methylthio)phenyl]-5-oxo-N-phenyl-1,4,5,6,7,8-hexahydro-3-quinolinecarboxamide,4-(isopropoxycarbonyl)benzyl 2-pyrazinecarboxylate,N-[4-(2-oxo-1-pyrrolidinyl)phenyl]-1H-1,2,4-triazole-3-carboxamide,4-chloro-N-(4-oxo-1,4-dihydro-2-quinazolinyl)benzamide,3-hydroxy-5-(4-propoxyphenyl)-1-(3-pyridinylmethyl)-4-(2-thienylcarbonyl)-1,5-dihydro-2H-pyrrol-2-one,4-(3,4-dimethoxyphenyl)-2-hydrazino-6-phenylpyrimidine,N′-[1-(3,4-dimethoxyphenyl)ethylidene]-3-phenyl-1H-pyrazole-5-carbohydrazide,3-[4-(4-chlorophenyl)-1-piperazinyl]-1-(4-iodaphenyl)-2,5-pyrrolidinedione,N-(3-oxo-1,3-dihydro-2-benzofuran-5-yl)-1H-1,2,4-triazole-3-carboxamide,3-[(5-methyl-2-furoyl)amino]benzoicacid,N-(4-{[(2,4-dimethoxyphenyl)amino]sulfonyl}phenyl)-3-[(4-methylphenyl)thio]propenamide,N~2~-(3-fluorophenyl)N~2~-(methylsulfonyl)N~1~-[2-(1-pyrrolidinylcarbonyl)phenyl]glycinamide,N-(5-chloro-2-methoxyphenyl)-N′-(l-ethyl-3,5-dimethyl-1H-pyrazol-4-yl)thiourea,1-[(4-methylphenyl)sulfonyl]N-1,3-thiazol-2-ylprolinamide,2,5-dichloro-N-(2-furylmethyl)benzamide, and 3-{[(2-methoxyphenyl)amino]methyl}-5-[4-(methylthio)benzylidene]-1,3-thiazolidine-2,4-dione,5-(4-propoxybenzyl)-1H-tetrazole, or a salt or non-salt form thereof.37. The method of claim 21, wherein the bacterial persister cells and/orVBNC cells are reduced in the bacterial population.
 38. The method ofclaim 21, wherein the bacterial persister cells and/or VBNC cells areeradicated from the bacterial population.
 39. The method of claim 21,wherein a reduction in the bacterial persister cells and/or the VBNCcells in the population occurs after the compound is administered to thepopulation.
 40. The method of claim 38, wherein the reduction of thepersister cells and/or the VBNC cells is greater than a reference,wherein the reference comprises a value obtained from reducing persistercells and/or or reducing VBNC cells of the same bacterial species usinga corresponding amount of an antibiotic which does not comprise anycompound of [5-nitro-3-phenyl-1H-indol-2-yl)methyl]amine hydrochloride,[2-({2-[4-chlorophenyl)amino]4-quinazolinyl}amino) ethanolhydrochloride,1-[2-(4-chlorophenoxy)-2-methylpropanoyl]-4-methylpiperazine,N-benzyl-N-{[3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl]methyl}acetamide,N-(3,4-dichlorophenyl)-N′-(3-fluorophenyl)thiourea,2-({2-[(4-bromophenyl)amino]-4-quinazolinyl}amino)ethanol hydrochloride,N-2-(4-ethoxyphenyl)-2,4-quinazolinediamine hydrochloride,4-{[(5-nitro-2-thienyl)methylene]amino}benzamide,2-[(6-phenyl-2,3,4,9-tetrahydro-1H-carbazol-1-yl)amino]ethanol,2-bromo-4-chlorophenyl phenylcarbamate,1-(3,6-dichloro-9H-carbazol-9-yl)-3-(2-methyl-1H-imidazol-1-yl)-2-propanol,N-(3-chloro-4-fluorophenyl)-N′-[2-(difluoromethoxy)phenyl]thiourea,2-(butyryloxy)-1H-benzo[de]isoquinoline-1,3(2H)-dione,N-phenyl-N′-[(1-phenylcyclopentyl)methyl]thiourea,N-[2-(4-fluorophenyl)ethyl]-N′-(4-nitrophenyl)thiourea,2,4-dichloro-5-(5-nitro-2-furyl)benzoic acid,N-(3-chlorophenyl)-N′-[3-(trifluoromethyl)phenyl]thiourea,N(3-chloro-4-fluorophenyl)-N′-(2-methoxy-4-nitrophenyl)thiourea, 4-({[3-chloro-4-fluorophenyl]amino}carbonothioyl)amino-N-ethylbenzenesulfonamide,2-[({2-[(2-chlorobenzyl)oxy]-1-naphthyl }methyl)amino]ethanolhydrochloride,17-(4-bromophenyl)-17-azapentacyclononadeca-2,4,6,9,11,13-hexaene-16,18-dione,N(3-chloro-4-fluorophenyl)-N′-3-pyridinylthiourea,N(4-chlorobenzyl)-N′-4-pyridinylthiourea,5-bromo-N-{2-[(4-methylphenyl)thio]ethyl}-2-thiophenesulfonamide,N′-(3,5-dichloro-2-hydroxybenzylidene)-2-oxo-4-phenyl-3-pyrrolidinecarbohydrazide,2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one,methyl5-[(dimethylamino)carbonyl]-4-methyl-2-({[(1-methyl-1H-pyrazol-3-yl)amino]carbonothioyl}amino)-3-thiophenecarboxylate,N-[2-(3,4-dimethoxyphenyl)ethyl]N′-[1-(pentafluorobenzyl)-1H-pyrazol-3-yl]thiourea,4-chloro-N-(6,7-dimethoxy-4-oxo-1,4-dihydro-2-quinazolinyl)benzamide,(4-methoxyphenyl)(phenyl) methanone,N-(3-acetylphenyl)-4,5-dimethyl-2-furamide,6-(4-iodophenyl)-2-methylimidazo[2,1-b][1,3]thiazole,N-{[(4-bromophenyl)amino]carbonothioyl}-2,2-dimethylpropanamide,1-(2,4-dichlorobenzoyl)-2,3-dihydro-1H-imidazo[1,2-a]benzimidazole,3-(3-chlorophenyl)-5,5-dimethyl-4-methylene-1,3-oxazolidin-2-one,2-methyl-4-[4-(methylthio)phenyl]-5-oxo-N-phenyl-1,4,5,6,7,8-hexahydro-3-quinolinecarboxamide,4-(isopropoxycarbonyl)benzyl 2-pyrazinecarboxylate,N-[4-(2-oxo-1-pyrrolidinyl)phenyl]-1H-1,2,4-triazole-3-carboxamide,4-chloro-N-(4-oxo-1,4-dihydro-2-quinazolinyl)benzamide,3-hydroxy-5-(4-propoxyphenyl)-1-(3-pyridinylmethyl)-4-(2-thienylcarbonyl)-1,5-dihydro-2H-pyrrol-2-one,4-(3,4-dimethoxyphenyl)-2-hydrazino-6-phenylpyrimidine,N′-[1-(3,4-dimethoxyphenyl)ethylidene]-3-phenyl-1H-pyrazole-5-carbohydrazide,3-[4-(4-chlorophenyl)-1-piperazinyl]-1-(4-iodophenyl)-2,5-pyrrolidinedione,N-(3-oxo-1,3-dihydro-2-benzofuran-5-yl)-1H-1,2,4-triazole-3-carboxamide,3-[(5-methyl-2-furoyl)amino]benzoicacid,N-(4-{[(2,4-dimethoxyphenyl)amino]sulfonyl}phenyl)-3-[(4-methylphenyl)thio]propenamide,N~2∼-(3-fluorophenyl)N~2~-(methylsulfonyl)N~1~-[2-(1-pyrrolidinylcarbonyl)phenyl]glycinamide,N-(5-chloro-2-methoxyphenyl)-N′-(1-ethyl-3,5-dimethyl-1H-pyrazol-4-yl)thiourea,1-[(4-methylphenyl)sulfonyl]N-1,3-thiazol-2-ylprolinamide,2,5-dichloro-N-(2-furylmethyl)benzamide, and3-{[(2-methoxyphenyl)amino]methyl}-5-[4-(methylthio)benzylidene]-1,3-thiazolidine-2,4-dione,5-(4-propoxybenzyl)-1H-tetrazole.